miR-29 mimics and uses thereof

ABSTRACT

The present invention relates to synthetic oligonucleotide mimetics of miRNAs. In particular, the present invention provides double-stranded, chemically-modified oligonucleotide mimetics of miR-29. Pharmaceutical compositions comprising the mimetics and their use in treating or preventing conditions associated with dysregulation of extracellular matrix genes, such as tissue fibrotic conditions, are also described.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/848,085, filed Sep. 8, 2015, now issued as U.S. Pat. No. 9,376,681,which claims the benefit of priority to U.S. Provisional Application No.62/047,562, filed on Sep. 8, 2014, the contents of which are herebyincorporated by reference in their entirety.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename:MIRG-047-02US_SeqList_ST25.txt, date recorded: Jun. 7, 2016, file size44 kilobytes).

FIELD OF THE INVENTION

The present invention relates to synthetic miRNA mimics or promiRs thatincrease miRNA activity in vivo. In particular, the present inventionrelates to mimics of miR-29 and their use in reducing collagendeposition and associated conditions, such as fibrosis.

BACKGROUND OF THE INVENTION

Based on gain- or loss-of-function data collected in animal diseasemodels using genetics or pharmacological modulation of microRNAs(miRNAs), it is now well accepted that miRNAs are important playersduring disease. These studies, combined with recent positive clinicalefficacy data (Janssen et al, 2013), underscore the relevance of miRNAsand the viability for miRNAs to become the next class of therapeutics.Indeed, miRNAs have several advantages as therapeutic interventionpoints in that they are small and comprise a known sequence.Additionally, since a single miRNA can regulate numerous target mRNAswithin biological pathways, modulation of a miRNA in principle allowsfor influencing an entire gene network and modifying complex diseasephenotypes (van Rooij & Olson, 2012).

While many studies have shown therapeutic efficacy using single-strandedmiRNA inhibitors called antimiRs, efforts to restore or increase thefunction of a miRNA have been lagging behind (van Rooij et al, 2012).Currently, miRNA function can be increased either by viraloverexpression or by using synthetic double-stranded miRNAs. So far theuse of adeno-associated viruses (AAV) to drive expression of a givenmiRNA for restoring its activity in vivo has shown to be effective in amouse model of hepatocellular and lung carcinoma (Kasinski & Slack,2012; Kota et al, 2009) and spinal and bulbar muscular atrophy (Miyazakiet al, 2012), while the use of unformulated syntheticoligonucleotide-based approaches to increase miRNA levels has not beenwell explored.

The microRNA-29 (miR-29) family is well characterized for their abilityto regulate extracellular matrix proteins (He et al, 2013). The familyconsists of miR-29a, -29b and -29c, which are expressed as twobicistronic clusters (miR-29a/-29b-1 and miR-29b-2/-29c), and arelargely homologous in sequence with only a few mismatches between thedifferent members in the 3′ regions of the mature miRNA (van Rooij etal, 2008). All three members are reduced in different types of tissuefibrosis and therapeutic benefit of increasing miR-29 levels has beenshown for heart (van Rooij et al, 2008), kidney (Qin et al, 2011; Wanget al, 2012; Xiao et al, 2012), liver (Roderburg et al, 2011; Sekiya etal, 2011; Zhang et al, 2012), lung (Cushing et al, 2011; Xiao et al,2012) and systemic sclerosis (Maurer et al, 2010).

There is a need in the art for synthetic oligonucleotide mimics ofmiR-29 that can effectively increase miR-29 activity in vivo. SuchmiR-29 mimics or miR-29 promiRs are useful for treating various tissuefibrotic conditions.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the discovery that miRNAmimics with modifications for stability and cellular uptake can be usedto replicate endogenous functions of miR-29. For instance, therapeutictreatment with a miR-29b mimic in the setting of pulmonary fibrosisrestores the bleomycin-induced reduction of miR-29 and blocks andreverses pulmonary fibrosis, which coincides with a repression of miR-29target genes that are induced during the disease process. Similarly,treatment of skin incisions with a miR-29b mimic down-regulates theexpression of extra-cellular matrix genes and other genes involved inthe fibrotic process. Accordingly, the present invention providesdouble-stranded RNA miR-29 mimetic compounds.

In some embodiments, the miR-29 mimetic compound comprises (a) a firststrand of about 23 to about 26 ribonucleotides comprising a maturemiR-29a, miR-29b, or miR-29c sequence; and (b) a second strand of about22 to about 23 ribonucleotides comprising a sequence that issubstantially complementary to the first strand, wherein the firststrand has a 3′ nucleotide overhang relative to the second strand. Incertain embodiments, the first strand and second strand contain one ormore modified nucleotides. The modified nucleotides may be 2′ sugarmodifications, such as 2′-alkyl (2′-O-methyl) or 2′-fluoromodifications. In one embodiment, the first strand has one or more2′-fluoro modifications. In another embodiment, the second strand hasone or more 2′-O-methyl modifications. In some embodiments, the secondstrand has one, two, three, or more mismatches relative to the firststrand. In one embodiment, the second strand of a miR-29 mimeticcompound contains mismatches at positions 4, 13, and/or 16 from the 3′end (of the second strand) relative to the first strand.

In one embodiment, the second strand of the miR-29 mimetic compound islinked to a cholesterol molecule at its 3′ terminus. In certainembodiments, the cholesterol molecule is linked to the second strandthrough at least a six carbon linker. The linker may be a cleavablelinker.

In one embodiment, the nucleotides comprising the 3′ overhang in thefirst strand are linked by phosphorothioate linkages.

In one embodiment, the miR-29 mimetic compound comprises a first strandcomprising the sequence of SEQ ID NO: 27 and a second strand comprisingthe sequence of SEQ ID NO: 5.

In another embodiment, the miR-29 mimetic compound comprises a firststrand comprising the sequence of SEQ ID NO: 19 and a second strandcomprising the sequence of SEQ ID NO: 1.

In yet another embodiment, the miR-29 mimetic compound comprises a firststrand comprising the sequence of SEQ ID NO: 19 and a second strandcomprising the sequence of SEQ ID NO: 15.

In yet another embodiment, the miR-29 mimetic compound comprises a firststrand comprising the sequence of SEQ ID NO: 33 and a second strandcomprising the sequence of SEQ ID NO: 1.

In yet another embodiment, the miR-29 mimetic compound comprises a firststrand comprising the sequence of SEQ ID NO: 34 and a second strandcomprising the sequence of SEQ ID NO: 1.

In yet another embodiment, the miR-29 mimetic compound comprises a firststrand comprising the sequence of SEQ ID NO: 35 and a second strandcomprising the sequence of SEQ ID NO: 24.

The present invention provides a pharmaceutical composition comprisingan effective amount of the miR-29 mimetic compounds described herein ora pharmaceutically-acceptable salt thereof, and apharmaceutically-acceptable carrier or diluent. In certain embodiments,the pharmaceutical composition is formulated for pulmonary, nasal,intranasal or ocular delivery and can be in the form of powders, aqueoussolutions, aqueous aerosols, nasal drops, aerosols, and/or ocular drops.In some embodiments, the pharmaceutical composition is administered withan inhalation system such as a nebulizer, a metered dose inhaler, a drypowder inhaler, or a soft mist inhaler.

The present invention also includes a method of regulating anextracellular matrix gene in a cell comprising contacting the cell withthe miR-29 mimetic compounds described herein. In one embodiment, theexpression or activity of the extracellular matrix gene is reducedfollowing contact with the miR-29 mimetic compound or composition. Insome embodiments, the extracellular matrix gene is a collagen gene, suchas Col1a1 and Col3a1. The cell may be in vitro, in vivo, or ex vivo.

The present invention provides a method of treating or preventing tissuefibrosis in a subject in need thereof. In one embodiment, the methodcomprises administering to the subject a miR-29 mimetic compounddescribed herein. In certain embodiments, the tissue fibrosis is cardiacfibrosis, pulmonary fibrosis, renal fibrosis, hepatic fibrosis, ocularfibrosis, cutaneous fibrosis including hypertrophic scarring andkeloids, hand, joint or tendon fibrosis, Peyronie's disease orscleroderma. In one embodiment, the tissue fibrosis is idiopathicpulmonary fibrosis (IPF).

In certain embodiments, the method for treating or preventing tissuefibrosis comprises administering a miR-29 mimetic compound or acomposition described herein via the pulmonary, nasal, or intranasalroute. In one embodiment, the miR-29 mimetic compound or the compositionis delivered via inhalation.

The present invention also includes a method of regulatingnon-extracellular matrix genes in a cell comprising contacting the cellwith the miR-29 mimetic compounds described herein. In one embodiment,the expression or activity of the non-extracellular matrix gene isincreased following contact with the miR-29 mimetic compound orcomposition. In some embodiments, the non-extracellular matrix gene is agene such as Itga3 or Numb. The cell may be in vitro, in vivo, or exvivo.

The present invention also provides a method for assessing the efficacyof a treatment with a miR-29 agonist or miR-29 antagonist, the methodcomprising determining a level of expression of one or more genes incells or a fibrotic tissue of a subject prior to the treatment with themiR-29 agonist or miR-29 antagonist, wherein the one or more genes areselected from a set of genes modulated by miR-29; determining the levelof expression of the same one or more genes in cells/fibrotic tissue ofthe subject after treatment with the miR-29 agonist or miR-29antagonist; and determining the treatment to be effective, lesseffective, or not effective based on the expression levels prior to andafter the treatment. In one embodiment, the one or more genes modulatedby miR-29 are selected from Table 5.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A. The double-stranded miR-29 mimics design contains a ‘guidestrand’ or ‘antisense strand’ that is identical to the miR-29b, with aUU overhang on the 3′ end, modified to increase stability, andchemically phosphorylated on the 5′ end and a ‘passenger strand’ or‘sense strand’ that contains 2′-O-Me modifications to prevent loadinginto RNA-induced silencing complex (RISC) as well as increase stabilityand is linked to cholesterol for enhanced cellular uptake. Severalmismatches are introduced in the sense strand to prevent this strandfrom functioning as an antimiR.

FIG. 1B. Transfection experiments in NIH 3T3 show a dose-dependentdecrease in Col1a1 with increasing amount of miR-29b mimic compared toeither untreated or mock treated cells. An siRNA directly targetingCol1a1 was taken along as a positive control. * p<0.05 versus mock, #p<0.05 versus untreated.

FIG. 1C. Northern blot analysis for miR-29b in different tissues 4 daysafter intravenous injection with 10, 50, 100, or 125 mpk miR-29b mimicindicates delivery to all tissues at the highest dose, with the mosteffective delivery taking place to the lungs and spleen compared tosaline injected mice. U6 is used as a loading control.

FIG. 1D. Real-time quantification of miR-29b mimicry indicates anincreased level of miR-29b at the higher dose levels with the mostefficient delivery to the lungs and spleen (n=4 per group). *p<0.05versus Saline injected animals.

FIG. 1E. Northern blot analysis for miR-29b in different tissues 1, 2, 4and 7 days after intravenous injection with 125 mpk of mimic indicatesthe presence of miR-29b mimic in all tissues examined, with a longerdetection in lung and spleen. U6 is used as a loading control.

FIG. 1F. Real-time quantification of miR-29b mimicry indicates anincreased level of miR-29b in all tissues measured which is maintainedthe longest in lungs and spleen (n=4 per group). *p<0.05 versus Salineinjected animals.

FIG. 2A. Real-time PCR analysis indicates a reduction in all miR-29family members in response to bleomycin, while miR-29 mimic treatmentresulted in the increased detection of miR-29b levels compared to eithercontrol or saline injected animals. *p<0.05 vs Saline/Saline

FIG. 2B. Real-time PCR analysis indicated a comparable decline in miR-29levels in pulmonary biopsies of patients with idiopathic pulmonaryfibrosis (IPF) compared to normal controls. *p<0.05 vs Normal

FIG. 2C. Histological examination by trichrome staining showingpronounced fibrotic and inflammatory response in response to bleomycin,which was blunted by miR-29b mimic treatment. Scale bar indicates 100μm.

FIG. 2D. Hydroxyproline measurements to assay for total collagen contentshowed a significant increase following bleomycin treatment in bothsaline and control treated groups, while there was no statisticaldifference in the miR-29 mimic treated group between Saline andbleomycin treated mice.

FIG. 2E-G. Cytokine measurements on bronchoalveolar lavage (BAL) fluidsindicated a significantly higher concentrations of IL-12, IL-4 and G-CSFwere detectable in BAL fluids from lungs from bleomycin treated mice,which was reduced with miR-29b mimic. (n=4), *p<0.05.

FIG. 2H. Bleomycin treatment increases the detection of immune cells inBAL fluids which was significantly reduced in the presence of miR-29bmimic while the Control mimic had no effect. (n=4), * p<0.05 vsSaline/Bleo, ^p<0.05 vs Control/Bleo

FIG. 3A-B. Bleomycin treatment increases the expression of Col1a1 andCol3a1 and the presence of miR-29b mimic inhibits Col1a1 and Col3a1 asmeasured by real-time PCR. MiR-29b mimicry has no effect on targetrepression under baseline conditions. (n=6-8), * p<0.05

FIG. 3C. IGF1 levels in BAL fluids increase following bleomycintreatment which were significantly blunted in the presence of miR-29mimic compared to both Saline and Control mimic treated mice. (n=4), *p<0.05.

FIG. 3D. Immunohistochemistry demonstrated robust detection of IGF1after bleomycin treatment, which was reduced in the miR-29b mimictreated group compared to Saline or Control mimic treated mice. Scalebar indicates 50 μm.

FIG. 4A. Hydroxyproline assessment showed a significant increasefollowing bleomycin treatment in both saline and control treated groups,however, there was no statistical difference in the miR-29 mimic treatedgroup between Saline and bleomycin treated mice. *P<0.05 (n=8)

FIG. 4B-C. Real-time PCR analysis for Col1a1 (B) and Col3a1 (C) showed asignificant increase with bleomycin treatment. miR-29b mimic treatmentnormalized both Col1a1 and Col3a1 to vehicle treated expression levels.*P<0.05 (n=8)

FIG. 4D. Histological examination by trichrome staining showing robustfibrosis in response to bleomycin, which was blunted by miR-29b mimictreatment.

FIG. 4E-F. Primary pulmonary fibroblasts from patients with IPF weretreated with vehicle or TGF-β and transfected with control mimic ormiR-29b mimic. Real-time PCR was performed for Col1a1 (E) and Col3a1(F). miR-29b mimic treatment showed a dose-dependent reduction in bothcollagens.

FIG. 4G-H. A549 cells were treated with vehicle or TGF-β and transfectedwith control mimic or miR-29b mimic. Real-time PCR was performed forCol1a1 (G) and Col3a1 (H). miR-29b mimic treatment showed adose-dependent reduction in expression of both Col1a1 and Col3a1.

FIG. 5. miR-29b mimic does not induce general signs of toxicity. MiR-29bmimic treatment does not induce any overt signs of liver or kidneytoxicity as indicated by the lack of change in aspartate or alaninetransaminases (AST and ALT). n=4 per group.

FIG. 6A. Increasing doses of miR-29b mimic fail to induce overt changesin gene expression under baseline conditions. Real-time PCR analysisindicates there to be no significant changes in expression in thedifferent tissue 4 days after treatment with increasing doses of miR-29bmimic for Col1a1 compared to Saline injected mice. n=4 per group, *p<0.05 compared to Saline injected.

FIG. 6B. Increasing doses of miR-29b mimic fail to induce overt changesin gene expression under baseline conditions. Real-time PCR analysisindicates there to be no significant changes in expression in thedifferent tissue 4 days after treatment with increasing doses of miR-29bmimic for Col3a1 compared to Saline injected mice. n=4 per group, *p<0.05 compared to Saline injected.

FIG. 7A. miR-29b mimic specifically increases miR-29b. MiR-29b mimicryspecifically increases the level of miR-29b without affecting the levelof miR-29a compared to Saline injected mice. n=4 per group.

FIG. 7B. miR-29b mimic specifically increases miR-29b. MiR-29b mimicryspecifically increases the level of miR-29b without affecting the levelof miR-29c compared to Saline injected mice. The increase in miR-29c atday 1 might be due to some cross-reactivity of the real-time probe. n=4per group.

FIG. 8A. miR-29b mimic does not induce any target changes in baselineconditions. Real-time PCR analysis showed the absence of significanttarget changes at the indicated timepoints after injecting 125 mpk ofmiR-29b mimic for Col1a1 compared to Saline injected mice. n=4 pergroup.

FIG. 8B. miR-29b mimic does not induce any target changes in baselineconditions. Real-time PCR analysis showed the absence of significanttarget changes at the indicated timepoints after injecting 125 mpk ofmiR-29b mimic for Col3a1 compared to Saline injected mice. n=4 pergroup.

FIG. 9. miR-29b mimic effects on gene expression in RAW cells. Real-timePCR analysis showed significant increases in Csf3, Igf1, and Kcexpression after miR-29b mimic treatment compared to vehicle or controlmimic. * p<0.05 compared to Vehicle injected.

FIG. 10A. miR29b mimic and antimiR effects on gene expression in mouseskin. A heatmap of the microarray data is presented, where upregulatedgenes are represented in red and downregulated genes are represented inblue, and the intensity of the color is representative of the foldchange relative to PBS control. The top portion of the heatmap (finelydashed box) contains genes which are repressed by miR-29b mimictreatment and upregulated by antimiR-29 treatment, and the bottomportion of the heatmap (wide dashed box) contains genes which areupregulated by miR-29b mimic treatment and repressed by antimiR-29treatment. All fold change and significance values are presented inTable 5.

FIG. 10B. miR29b mimic and antimiR effects on gene expression in mouseskin. DAVID analysis (NCBI) of functional terms that are enriched in thetwo groups shown in FIG. 10A are presented. Gene Ontology (GO) terms ofExtracellular Matrix, (Skin) Function, Adhesion/Cell Signaling and CellDifferentiation/Apoptosis are the top negatively regulated pathwaysfollowing miR-29b mimic treatment and Cellular (Nuclear) Structure andRNA Processing are the top positively regulated pathways followingmiR-29b mimic treatment. The microarray analysis of skin and acute skinwounds in C57BL/6 mice shows reciprocal regulation of 228 genes byintradermal treatment with a miR-29b mimic comprising SEQ ID NO: 2 andSEQ ID NO: 1, and antimiR-29 (SEQ ID NO: 36).

FIG. 11A. Quantitative real-time (RT)-PCR analysis of mouse incisionalwounds treated with intradermal miR-29b mimic (SEQ ID NO: 2/SEQ IDNO: 1) or PBS control. Data are presented as a grouped bar graph wherethe first bar in each treatment group represents the expression level ofthe first gene in the list on the right, and so on. RT-PCR confirmedthat collagens, other extracellular matrix genes and other direct andindirect target genes previously shown to be repressed by miR-29b mimictreatment (FIG. 10a , upper half of the heatmap) show a dose-dependentreduction in expression with miR-29b mimic treatment in acute skinwounds. **** p<0.0001 versus PBS treated incisions, using a 2-way ANOVAwith treatment and gene as the two factors assessed.

FIG. 11B. Genes previously identified as being de-repressed with miR-29bmimic treatment (FIG. 10A, lower half of the heatmap) show adose-dependent increase in expression with miR-29b mimic treatment byquantitative RT-PCR. *** p<0.001 and **** p<0.0001 versus PBS treatedincisions, using a 2-way ANOVA with treatment and gene as the twofactors assessed. The miR-29b mimic significantly affects the expressionof miR-29 target genes in acute skin wounds.

FIG. 12. Activity of miR-29 mimics and effects of nucleotidemodifications. Transfection experiments in IMR-90 human lung fibroblastsshow that different miR-29 mimics have different levels of activity asmeasured by repression of collagen gene expression. * p<0.05, ***p<0.001 and **** p<0.0001 versus mock transfection, using a 2-way ANOVAwith treatment and gene as the two factors assessed.

FIG. 13. In vivo activity of miR-29b mimics with linker modifications.Mice with incisional wounds were treated with 20 nmol of various miR-29bmimics that differ only in the linkage between the cholesterol moietyand the second/sense strand. Activity of the miR-29b mimics wasdetermined by measuring the expression of five collagen synthesis genes.

FIG. 14. Effect of 5′ phosphorylation on the activity of miR-29b mimics.RAB-9 skin fibroblasts were transfected with varying concentrations ofmiR-29b mimics with (SEQ ID NO: 2/SEQ ID NO: 1) and without (SEQ ID NO:19/SEQ ID NO: 1) 5′ phosphorylation on the antisense strand. Activity ofthe miR-29b mimics was determined by measuring the expression of threecollagen synthesis genes.

DETAILED DESCRIPTION OF THE INVENTION

Over the last decade great enthusiasm has evolved for microRNA (miRNA)therapeutics. Part of the excitement stems from the fact that a miRNAoften regulates numerous related mRNAs. As such, modulation of a singlemiRNA allows for parallel regulation of multiple genes involved in aparticular disease. While many studies have shown therapeutic efficacyusing miRNA inhibitors, efforts to restore or increase the function of amiRNA have been lagging behind.

The miR-29 family has gained a lot of attention for its clear functionin tissue fibrosis. This fibroblast-enriched miRNA family isdownregulated in fibrotic diseases which induces a coordinate increaseof many extracellular matrix genes. The present inventors have foundthat administration of synthetic RNA duplexes can increase miR-29 levelsin vivo for several days. Moreover, therapeutic delivery of these miR-29mimics during bleomycin-induced pulmonary fibrosis restores endogenousmiR-29 function thereby decreasing collagen expression and blocking andreversing pulmonary fibrosis. Furthermore, administration of miR-29mimics of the present invention to skin incisions downregulatesextracellular matrix genes and other genes involved in the fibroticprocess. These data support the feasibility of designing effective miRNAmimics to therapeutically increase miRNAs and indicate miR-29 to be apotent therapeutic miRNA for treating various fibrotic conditions anddisorders involving increased collagen production. Accordingly, thepresent invention provides miR-29 mimics, compositions and uses thereof.

A microRNA mimetic compound according to the invention comprises a firststrand and a second strand, wherein the first strand comprises a maturemiR-29a, miR-29b, or miR-29c sequence and the second strand comprises asequence that is substantially complementary to the first strand and hasat least one modified nucleotide. Throughout the disclosure, the term“microRNA mimetic compound” may be used interchangeably with the terms“promiR-29,” “miR-29 agonist,” “microRNA agonist,” “microRNA mimic,”“miRNA mimic,” or “miR-29 mimic;” the term “first strand” may be usedinterchangeably with the terms “antisense strand” or “guide strand”; theterm “second strand” may be used interchangeably with the term “sensestrand” or “passenger strand;” and the term “miR-29 antagonist” may beused interchangeably with the terms “oligonucleotide inhibitor,”“antimiR-29,” “antisense oligonucleotide,” “miR-29 antagomir” or“anti-microRNA oligonucleotide.”

In one embodiment, the first strand of the microRNA mimetic compoundcomprises from about 23 to about 26 nucleotides comprising a sequence ofmature miR-29a, miR-29b, or miR-29c and the second strand comprises fromabout 22 to about 23 nucleotides comprising a sequence that ispartially, substantially, or fully complementary to the first strand. Invarious embodiments, the first strand may comprise about 23, 24, 25, or26 nucleotides and the second strand may comprise about 22 or 23nucleotides.

The nucleotides that form the first and the second strand of themicroRNA mimetic compounds may comprise ribonucleotides,deoxyribonucleotides, modified nucleotides, and combinations thereof. Incertain embodiments, the first strand and the second strand of themicroRNA mimetic compound comprise ribonucleotides and/or modifiedribonucleotides. The term “modified nucleotide” means a nucleotide wherethe nucleobase and/or the sugar moiety is modified relative tounmodified nucleotides.

In certain embodiments, the microRNA mimetic compounds have a firststrand or an antisense strand, whose sequence is identical to all orpart of a mature miR-29a, miR-29b, or miR-29c sequence, and a secondstrand or a sense strand whose sequence is about 70% to about 100%complementary to the sequence of the first strand. In one embodiment,the first strand of the miRNA mimetic compound is at least about 75, 80,85, 90, 95, or 100% identical, including all integers there between, tothe entire sequence of a mature, naturally occurring miR-29a, miR-29b,or miR-29c sequence. In certain embodiments, the first strand is aboutor is at least about 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%identical to the sequence of a mature, naturally-occurring miRNA, suchas the mouse, human, or rat miR-29a, miR-29b, or miR-29c sequence.Alternatively, the first strand may comprise 20, 21, 22, or 23nucleotide positions in common with a mature, naturally-occurring miRNAas compared by sequence alignment algorithms and methods well known inthe art.

It is understood that the sequence of the first strand is considered tobe identical to the sequence of a mature miR-29a, miR-29b, or miR-29ceven if the first strand includes a modified nucleotide instead of anaturally-occurring nucleotide. For example, if a mature,naturally-occurring miRNA sequence comprises a cytidine nucleotide at aspecific position, the first strand of the mimetic compound may comprisea modified cytidine nucleotide, such as 2′-fluoro-cytidine, at thecorresponding position or if a mature, naturally-occurring miRNAsequence comprises a uridine nucleotide at a specific position, themiRNA region of the first strand of the mimetic compound may comprise amodified uridine nucleotide, such as 2′-fluoro-uridine,2′-O-methyl-uridine, 5-fluorouracil, or 4-thiouracil at thecorresponding position. Thus, as long as the modified nucleotide has thesame base-pairing capability as the nucleotide present in the mature,naturally-occurring miRNA sequence, the sequence of the first strand isconsidered to be identical to the mature, naturally-occurring miRNAsequence. In some embodiments, the first strand may include amodification of the 5′-terminal residue. For example, the first strandmay have a 5′-terminal monophosphate. In some other embodiments, thefirst strand does not contain a 5′-terminal monophosphate.

In some embodiments, the second strand of the microRNA mimic ispartially complementary to the sequence of the first strand. Forexample, the sequence of the second strand is at least about 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, inclusive of all valuestherebetween, complementary to the sequence of the first strand. In someother embodiments, the second strand is substantially complementary tothe sequence of the first strand. For example, the second strand is atleast about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99%, inclusive of all values therebetween, complementary tothe sequence of the first strand. In yet some other embodiments, thesequence of the second strand may be fully complementary to the firststrand. In certain embodiments, about 19, 20, 21, 22, or 23 nucleotidesof the complementary region of the second strand may be complementary tothe first strand.

It is understood that the sequence of the second strand is considered tobe complementary to the first strand even if the second strand includesa modified nucleotide instead of a naturally-occurring nucleotide. Forexample, if the first strand sequence comprises a guanosine nucleotideat a specific position, the second strand may comprise a modifiedcytidine nucleotide, such as 2′-O-methyl-cytidine, at the correspondingposition.

In some embodiments, the second strand comprises about 1, 2, 3, 4, 5, or6 mismatches relative to the first strand. That is, up to 1, 2, 3, 4, 5,or 6 nucleotides between the first strand and the second strand may notbe complementary. In one embodiment, the mismatches are not consecutiveand are distributed throughout the second strand. In another embodiment,the mismatches are consecutive and may create a bulge. In oneembodiment, the second strand contains 3 mismatches relative to thefirst strand. In certain embodiments, the second strand of a miR-29amimic or a miR-29c mimic contains mismatches at positions 4, 13, and/or16 from the 3′ end (of the second strand) relative to the first strand.In one embodiment, the second strand of a miR-29b mimic containsmismatches at positions 4, 13, and/or 16 from the 3′ end (of the secondstrand) relative to the first strand. In another embodiment, the secondstrand of a miR-29b mimic contains mismatches at positions 4, 9, 10, 11,13 and/or 16 from the 3′ end (of the second strand) relative to thefirst strand.

In some embodiments, the first and/or the second strand of the mimeticcompound may comprise an overhang on the 5′ or 3′ end of the strands. Incertain embodiments, the first strand comprises a 3′ overhang, i.e., asingle-stranded region that extends beyond the duplex region, relativeto the second strand. The 3′ overhang of the first strand may range fromabout one nucleotide to about four nucleotides. In certain embodiments,the 3′ overhang of the first strand may comprise 1 or 2 nucleotides. Insome embodiments, the nucleotides comprising the 3′ overhang in thefirst strand are linked by phosphorothioate linkages. The nucleotidescomprising the 3′ overhang in the first strand may includeribonucleotides, deoxyribonucleotides, modified nucleotides, orcombinations thereof. In certain embodiments, the 3′ overhang in thefirst strand comprises two ribonucleotides. In one embodiment, the 3′overhang of the first strand comprises two uridine nucleotides linkedthrough a phosphorothioate linkage. In some embodiments, the firststrand may not contain an overhang.

In one embodiment, the nucleotides in the second/sense strand of miR-29mimics of the invention are linked by phosphodiester linkages and thenucleotides in the first/antisense strand are linked by phosphodiesterlinkages except for the last three nucleotides at the 3′ end which arelinked to each other via phosphorothioate linkages.

In various embodiments, miR-29 mimics of the present invention comprisemodified nucleotides. For instance, in one embodiment, the first strandof the mimic comprises one or more 2′-fluoro nucleotides. In anotherembodiment, the first strand may not include any modified nucleotide. Inone embodiment, the second strand comprises one or more 2′-O-methylmodified nucleotides.

In various embodiments, miR-29 mimics according to the present inventioncomprise first and second strands listed in the Tables below.Definitions of the modifications are presented in Table 4. These miR-29mimetic compounds are useful for regulating the expression ofextracellular matrix genes in a cell and treating associated conditions,such as tissue fibrosis, dermal fibrosis, including the uses andconditions described in WO 2009/018493, which is hereby incorporated byreference in its entirety.

TABLE 1 miR-29a mimics SEQ ID Modified Sequence NO.Second/sense/passenger strands5′-mU.mA.rA.rC.rC.rG.rA.rU.rU.rU.rC.rA.rG.rA.rU.rG.rG.rU.rG.rC.rU.rA.rU.rU-3′ 35′-mU.mA.rA.mC.mC.rG.mU.mU.mU.rA.mC.rA.rG.rA.mU.rG.rG.mU.mC.mC.mU.rA-3′ 45′-mU.mA.rA.mC.mC.rG.mU.mU.mU.rA.mC.rA.rG.rA.mU.rG.rG.mU.mC.mC.mU.rA.chol6-3′ 55′-mU.mA.rA.rC.rC.rG.rA.rU.rU.rU.rC.rA.rG.rA.rU.rG.rG.rU.rG.rC.rU.rAs.rUs.rUs.chol6-3′115′-mU.mA.rA.rC.rC.rG.rA.rU.rU.rU.rC.rA.rG.rA.rU.rG.rG.rU.rG.rC.rU.rA-3′37 First/antisense/guide strands5′-p.rU.rA.rG.rC.rA.rC.rC.rA.rU.rC.rU.rG.rA.rA.rA.rU.rC.rG.rG.rU.rU.rA.rU.rU-3′ 65′-p.fU.rA.rG.fC.rA.fC.fC.rA.fU.fC.fU.rG.rA.rA.rA.fU.fC.rG.rG.fU.fU.rAs.rUs.rU-3′ 75′-fU.rA.rG.fC.rA.fC.fC.rA.fU.fC.fU.rG.rA.rA.rA.fU.fC.rG.rG.fU.fU.rAs.rUs.rU-3′275′-rU.rA.rG.rC.rA.rC.rC.rA.rU.rC.rU.rG.rA.rA.rA.rU.rC.rG.rG.rU.rU.rA.rU.rU-3′38

TABLE 2 miR-29b mimics SEQ ID Modified Sequence NO.Second/sense/passenger strands5′-mA.mA.rC.rA.rC.rU.rG.rA.rU.rU.rU.rC.rA.rA.rA.rU.rG.rG.rU.rG.rC.rU.rA.rU.rU-3′ 85′-mA.mA.mC.rA.mC.mU.rG.rA.mU.mU.mU.mC.rA.rA.rA.mU.rG.rG.mU.rG.mC.mU.rA.chol6-3′ 95′-mA.mA.rC.rA.rC.rU.rG.rA.rU.rU.rU.rC.rA.rA.rA.rU.rG.rG.rU.rG.rC.rU.rAs.rUs.rUs.chol6-3′105′-mA.mA.mC.rA.mC.mU.rG.mU.mU.mU.rA.mC.rG.rG.rG.mU.rG.rG.mU.mC.mC.mU.rA-3′135′-mA.mA.mC.rA.mC.mUsG.mU.mU.mU.rA.mC.rG.rG.rG.mU.rG.rG.mU.mC.mC.mUsA.chol6-3′145′-mA.mA.mC.rA.mC.mU.rG.mU.mU.mU.rA.mC.rA.rA.rA.mU.rG.rG.mU.mC.mC.mU.rA.chol6-3′ 1 5′- 15mA.mA.mC.rA.mC.mU.rG.mU.mU.mU.rA.mC.rA.rA.rA.mU.rG.rG.mU.mC.mC.mU.rA.dT.dT.chol6-3′5′- 16C6Chol.dT.dT.mA.mA.mC.rA.mC.mU.rG.mU.mU.mU.rA.mC.rA.rA.rA.mU.rG.rG.mU.mC.mC.mU.rA-3′5′-mA.mA.mC.rA.mC.mUsG.mU.mU.mU.rA.mC.rA.rA.rA.mU.rG.rG.mU.mC.mC.mUsA.chol9-3′175′-rA.mA.rC.mA.rC.mU.rG.mA.rU.mU.rU.mC.rA.mA.rA.mU.rG.mG.rU.mG.rC.mU.rA.chol6-3′285′-rA.mA.rC.mA.rC.mU.rG.mA.rU.mU.rU.mC.rA.mA.rA.mU.rG.mG.rU.mG.rC.mU.rAsrU.rUs.rU.chol6-3′295′-mA.mA.mC.rA.mC.mU.rG.mU.mU.mU.rA.mC.rA.rA.rA.mU.rG.rG.mU.mC.mC.mU.rA.cholTEG-3′305′-mA.mA.rC.rA.rC.rU.rG.rA.rU.rU.rU.rC.rA.rA.rA.rU.rG.rG.rU.rG.rC.rU.rA-3′39 First/antisense/guidestrands5′-p.rU.rA.rG.rC.rA.rC.rC.rA.rU.rU.rU.rG.rA.rA.rA.rU.rC.rA.rG.rU.rG.rU.rU.rU.rU-3′185′-p.fU.rA.rG.fC.rA.fC.fC.rA.fU.fU.fU.rG.rA.rA.rA.fU.fC.rA.rG.fU.rG.fU.fUs.rUs.rU-3′ 25′-fU.rA.rG.fC.rA.fC.fC.rA.fU.fU.fU.rG.rA.rA.rA.fU.fC.rA.rG.fU.rG.fU.fUs.rUs.rU-3′195′-p.fU.rA.rG.fC.rA.fC.fC.rA.fC.fC.fC.rG.rA.rA.rA.fU.fC.rA.rG.fU.rG.fU.fUs.rUs.rU-3′205′-rU.rA.rG.rC.rA.rC.rC.rA.rU.rU.rU.rG.rA.rA.rA.rU.rC.rA.rG.rU.rG.rU.rU.rU.rU-3′215′-mU.rA.mG.rC.mA.rC.mC.rA.mU.rU.mU.rG.mA.rA.mA.rU.mC.rA.mG.rU.mG.rU.mU-3′31 5′mU.rA.mG.rC.mA.rC.mC.rA.mU.rU.mU.rG.mA.rA.mA.rU.mC.rA.mG.rU.mG.rU.mUs.rUs.rU-3′325′-mU.rA.rG.mC.rA.mC.mC.rA.mU.mU.mU.rG.rA.rA.rA.mU.mC.rA.rG.mU.rG.mU.mUs.rUs.rU-3′335′-fU.rA.rG.fC.rA.fC.fC.rA.fU.fU.fU.rG.rA.rA.rA.fU.fC.rA.rG.fU.rG.fU.fU-3′345′-rU.rA.rG.rC.rA.rC.rC.rA.rU.rU.rU.rG.rA.rA.rA.rU.rC.rA.rG.rU.rG.rU.rU.rU.rU-3′40

TABLE 3 miR-29c mimics SEQ ID Modified Sequence NO.Second/sense/passenger strands5′-mU.mA.rA.rC.rC.rG.rA.rU.rU.rU.rC.rA.rA.rA.rU.rG.rG.rU.rG.rC.rU.rA.rU.rU-3′225′-mU.mA.rA.mC.mC.rG.mU.mU.mU.rA.mC.rA.rA.rA.mU.rG.rG.mU.mC.mC.mU.rA-3′235′-mU.mAsA.mC.mC.rG.mU.mU.mUsA.mC.rA.rA.rA.mUsG.rG.mU.mC.mC.mUsA.chol6-3′245′-mU.mA.rA.rC.rC.rG.rA.rU.rU.rU.rC.rA.rA.rA.rU.rG.rG.rU.rG.rC.rU.rAs.rUs.rUs.chol6-3′125′-mU.mA.rA.rC.rC.rG.rA.rU.rU.rU.rC.rA.rA.rA.rU.rG.rG.rU.rG.rC.rU.rA-3′41 First/antisense/guide strands5′-p.rU.rA.rG.rC.rA.rC.rC.rA.rU.rU.rU.rG.rA.rA.rA.rU.rC.rG.rG.rU.rU.rA.rU.rU-3′255′-p.fU.rA.rG.fC.rA.fC.fC.rA.fU.fU.fU.rG.rA.rA.rA.fU.fC.rG.rG.fU.fU.rAs.rUs.rU-3′265′-fU.rA.rG.fC.rA.fC.fC.rA.fU.fU.fU.rG.rA.rA.rA.fU.fC.rG.rG.fU.fU.rAs.rUs.rU-3′355′-rU.rA.rG.rC.rA.rC.rC.rA.rU.rU.rU.rG.rA.rA.rA.rU.rC.rG.rG.rU.rU.rA.rU.rU-3′42

TABLE 4 Definitions of Abbreviations Nucleotide unit or Nucleotide unitor modification Abbreviation modification Abbreviation ribo A rA ribo AP═S rAs ribo G rG ribo G P═S rGs ribo C rC ribo C P═S rCs ribo U rU riboU P═S rUs O-methyl A mA O-methyl A P═S mAs O-methyl G mG O-methyl G P═SmGs O-methyl C mC O-methyl C P═S mCs O-methyl U mU O-methyl U P═S mUsfluoro C fC fluoro C P═S fCs fluoro U fU fluoro U P═S fUs deoxy A dAdeoxy A P═S dAs deoxy G dG deoxy G P═S dGs deoxy C dC deoxy C P═S dCsdeoxy T dT deoxy T P═S dTs monophosphate p Cholesterol conjugateChol6/C6 chol with a 6 carbon linker Cholesterol conjugate Chol9 with a9 carbon linker

In certain embodiments, a miR-29a mimic comprises a first strandcomprising SEQ ID NO: 27 and a second strand comprising SEQ ID NO: 5. Inother embodiments, a miR-29a mimic comprises a first strand comprisingSEQ ID NO: 7 and a second strand comprising SEQ ID NO: 5.

In some embodiments, a miR-29b mimic comprises a first strand comprisingSEQ ID NO: 19 and a second strand comprising SEQ ID NO: 1. In some otherembodiments, a miR-29b mimic comprises a first strand comprising SEQ IDNO: 2 and a second strand comprising SEQ ID NO: 1. In yet some otherembodiments, a miR-29b mimic comprises a first strand comprising SEQ IDNO: 19 and a second strand comprising SEQ ID NO: 15. In yet some otherembodiments, a miR-29b mimic comprises a first strand comprising SEQ IDNO: 33 and a second strand comprising SEQ ID NO: 1. In yet some otherembodiments, a miR-29b mimic comprises a first strand comprising SEQ IDNO: 34 and a second strand comprising SEQ ID NO: 1. In yet some otherembodiments, a miR-29b mimic comprises a first strand comprising SEQ IDNO: 19 and a second strand comprising SEQ ID NO: 30.

In certain embodiments, a miR-29c mimic comprises a first strandcomprising SEQ ID NO: 35 and a second strand comprising SEQ ID NO: 24.In other embodiments, a miR-29c mimic comprises a first strandcomprising SEQ ID NO: 26 and a second strand comprising SEQ ID NO: 24.

The modified nucleotides that may be used in the microRNA mimeticcompounds of the invention can include nucleotides with a basemodification or substitution. The natural or unmodified bases in RNA arethe purine bases adenine (A) and guanine (G), and the pyrimidine basescytosine (C) and uracil (U) (DNA has thymine (T)). In contrast, modifiedbases, also referred to as heterocyclic base moieties, include othersynthetic and natural nucleobases such as 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine and other alkynyl derivatives of pyrimidine bases,6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil),4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl andother 8-substituted adenines and guanines, 5-halo (including 5-bromo,5-trifluoromethyl and other 5-substituted uracils and cytosines),7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine,8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and3-deazaguanine and 3-deazaadenine.

In some embodiments, the microRNA mimetic compounds can have nucleotideswith modified sugar moieties. Representative modified sugars includecarbocyclic or acyclic sugars, sugars having substituent groups at oneor more of their 2′, 3′ or 4′ positions and sugars having substituentsin place of one or more hydrogen atoms of the sugar. In certainembodiments, the sugar is modified by having a substituent group at the2′ position. In additional embodiments, the sugar is modified by havinga substituent group at the 3′ position. In other embodiments, the sugaris modified by having a substituent group at the 4′ position. It is alsocontemplated that a sugar may have a modification at more than one ofthose positions, or that an RNA molecule may have one or morenucleotides with a sugar modification at one position and also one ormore nucleotides with a sugar modification at a different position.

Sugar modifications contemplated in the miRNA mimetic compounds include,but are not limited to, a substituent group selected from: OH; F; O-,S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; orO-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may besubstituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl andalkynyl.

In some embodiments, miRNA mimetic compounds have a sugar substituentgroup selected from the following: C₁ to C₁₀ lower alkyl, substitutedlower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl,SH, SCH₃, Cl, Br, CN, OCN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, or similarsubstituents. In one embodiment, the modification includes2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, which is also known as2′-O-(2-methoxyethyl) or 2′-MOE), that is, an alkoxyalkoxy group.Another modification includes 2′-dimethylaminooxyethoxy, that is, aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE and2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), that is,2′-O—CH₂—O—CH₂—N(CH₃)₂.

Sugar substituent groups on the 2′ position (2′-) may be in the arabino(up) position or ribo (down) position. One 2′-arabino modification is2′-F. Other similar modifications may also be made at other positions onthe sugar moiety, particularly the 3′ position of the sugar on the 3′terminal nucleoside or in 2′-5′ linked oligonucleotides and the 5′position of 5′ terminal nucleotide.

In certain embodiments, the sugar modification is a 2′-O-alkyl (e.g.2′-O-methyl, 2′-O-methoxyethyl), 2′-halo (e.g., 2′-fluoro, 2′-chloro,2′-bromo), and 4′ thio modifications. For instance, in some embodiments,the first strand of the miR-29a, miR-29b, or miR-29c mimetic compoundcomprises one or more 2′ fluoro nucleotides. In another embodiment, thefirst strand of the mimetic compounds has no modified nucleotides. Inyet another embodiment, the second strand of miR-29a, miR-29b, ormiR-29c mimetic compound comprises one or more 2′-O-methyl modifiednucleotides.

The first and the second strand of microRNA mimetic compounds of theinvention can also include backbone modifications, such as one or morephosphorothioate, morpholino, or phosphonocarboxylate linkages (see, forexample, U.S. Pat. Nos. 6,693,187 and 7,067,641, which are hereinincorporated by reference in their entireties). For example, in someembodiments, the nucleotides comprising the 3′ overhang in the firststrand are linked by phosphorothioate linkages.

In some embodiments, the microRNA mimetic compounds are conjugated to acarrier molecule such as a steroid (cholesterol), a vitamin, a fattyacid, a carbohydrate or glycoside, a peptide, or other small moleculeligand to facilitate in vivo delivery and stability. Preferably, thecarrier molecule is attached to the second strand of the microRNAmimetic compound at its 3′ or 5′ end through a linker or a spacer group.In various embodiments, the carrier molecule is cholesterol, acholesterol derivative, cholic acid or a cholic acid derivative. The useof carrier molecules disclosed in U.S. Pat. No. 7,202,227, which isincorporated by reference herein in its entirety, is also envisioned. Incertain embodiments, the carrier molecule is cholesterol and it isattached to the 3′ or 5′ end of the second strand through at least a sixcarbon linker. In one embodiment, the carrier molecule is attached tothe 3′ end of the second strand through a six or nine carbon linker. Insome embodiments, the linker is a cleavable linker. In variousembodiments, the linker comprises a substantially linear hydrocarbonmoiety. The hydrocarbon moiety may comprise from about 3 to about 15carbon atoms and may be conjugated to cholesterol through a relativelynon-polar group such as an ether or a thioether linkage. In certainembodiments, the hydrocarbon linker/spacer comprises an optionallysubstituted C2 to C15 saturated or unsaturated hydrocarbon chain (e.g.alkylene or alkenylene). A variety of linker/spacer groups described inU.S. Pre-grant Publication No. 2012/0128761, which is incorporated byreference herein in its entirety, can be used in the present invention.

In various embodiments, the present invention provides methods oftreating, ameliorating, or preventing fibrotic conditions in a subjectin need thereof comprising administering to the subject atherapeutically effective amount of at least one of a miR-29a, miR-29b,and/or miR-29c mimic described herein. Fibrotic conditions that may betreated using miR-29 mimics of the invention include, but are notlimited to, tissue fibrosis such as pulmonary fibrosis, cardiacfibrosis, hepatic fibrosis, kidney fibrosis, diabetic fibrosis, skeletalmuscle fibrosis, and ocular fibrosis; and dermal/cutaneous fibrosis suchas keloids, cutaneous sclerosis, systemic sclerosis (scleroderma),hypertrophic scars, hand/joint/tendon fibrosis, and Peyronie's disease.In one embodiment, the fibrotic condition treated using the miR-29mimics of the invention is idiopathic pulmonary fibrosis. Use of miR-29agonists in treating certain fibrotic conditions is described in U.S.Pat. No. 8,440,636, which is hereby incorporated by reference herein.

In one embodiment, administration of miR-29 mimics of the presentinvention reduces the expression or activity one or more extracellularmatrix genes in cells of the subject. In another embodiment,administration of miR-29 mimics of the present invention reduces theexpression or activity one or more collagen synthesis genes in cells ofthe subject. In yet another embodiment, administration of miR-29 mimicsup-regulates the expression or activity one or more genes involved inthe skin development, epidermis development, ectoderm development andcellular homeostasis. Cells of the subject where the expression oractivity of various genes is regulated by miR-29 mimics of the inventioninclude fibroblasts and epidermal cells. In some embodiments,administration of miR-29 mimics down-regulates inflammatory responsesassociated with fibrosis. For example, administration of miR-29 mimicsreduces the levels of pro-inflammatory cytokines such as IL-12, IL-4,GCSF, and TNF-α in fibrosis patients. Administration of miR-29 mimicsmay also reduce infiltration of immune effector cells such asneutrophils, lymphocytes, monocytes, and macrophages in fibrotic tissuesor organs.

In certain embodiments, the present invention provides methods ofregulating an extracellular matrix gene in a cell comprising contactingthe cell with a miR-29 mimic of the present invention. In someembodiments, the invention provides methods of regulating a collagensynthesis gene in a cell comprising contacting the cell with a miR-29mimic of the present invention. Upon treatment or contact, the miR-29mimic reduces the expression or activity of the extracellular matrixgene or the collagen synthesis gene.

As used herein, the term “subject” or “patient” refers to any vertebrateincluding, without limitation, humans and other primates (e.g.,chimpanzees and other apes and monkey species), farm animals (e.g.,cattle, sheep, pigs, goats and horses), domestic mammals (e.g., dogs andcats), laboratory animals (e.g., rabbits, rodents such as mice, rats,and guinea pigs), and birds (e.g., domestic, wild and game birds such aschickens, turkeys and other gallinaceous birds, ducks, geese, and thelike). In some embodiments, the subject is a mammal. In otherembodiments, the subject is a human.

The invention also provides methods for assessing the efficacy of atreatment with miR-29 agonists (e.g. drugs or miR-29 mimics) or miR-29antagonists (e.g. drugs or antimiR-29). For instance, in one embodiment,the method for assessing the treatment efficacy comprises determining alevel of expression of one or more genes in cells or a fibrotic tissueof a subject prior to the treatment with miR-29 mimics or miR-29antagonists, wherein the one or more genes are selected from a set ofgenes modulated by miR-29, e.g. genes listed in Table 5; determining thelevel of expression of the same one or more genes in cells/fibrotictissue of the subject after treatment with miR-29 mimics or miR-29antagonist; and determining the treatment to be effective, lesseffective, or not effective based on the expression levels prior to andafter the treatment. That is, in one embodiment, the genes listed inTable 5 serve as a biomarker for clinical efficacy of the miR-29 mimicor miR-29 antagonist treatment. In one embodiment, a statisticallysignificant difference in the expression of the genes prior to and aftertreatment indicates the treatment to be effective. In anotherembodiment, at least 1-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold,3.5-fold, or 4-fold difference in the expression of the genes prior toand after treatment indicates the treatment to be effective.

The present invention also provides pharmaceutical compositionscomprising a therapeutically effective amount of one or more microRNAmimetic compounds of miR-29a, miR-29b, and/or miR-29c according to theinvention or a pharmaceutically acceptable salt thereof, and apharmaceutically acceptable carrier or excipient.

In one embodiment, the pharmaceutical composition comprises atherapeutically effective amount of a miR-29a mimetic compound and apharmaceutically acceptable carrier or excipient, wherein the firststrand of the mimetic compound comprises a mature miR-29a sequence andthe second strand is substantially complementary to the first strand. Inanother embodiment, the pharmaceutical composition comprises atherapeutically effective amount of a miR-29b mimetic compound and apharmaceutically acceptable carrier or excipient, wherein the firststrand of the mimetic compound comprises a mature miR-29b sequence andthe second strand is substantially complementary to the first strand. Inyet another embodiment, the pharmaceutical composition comprises atherapeutically effective amount of a miR-29c mimetic compound and apharmaceutically acceptable carrier or excipient, wherein the firststrand of the mimetic compound comprises a mature miR-29c sequence andthe second strand is substantially complementary to the first strand.

In some embodiments, the pharmaceutical composition comprises atherapeutically effective amount of at least two microRNA mimeticcompounds of the invention and a pharmaceutically acceptable carrier orexcipient. For instance, a pharmaceutical composition may comprise acombination of a miR-29a and a miR-29b mimics; a miR-29a and a miR-29cmimics; or a miR-29b and a miR-29c mimics. Alternatively, thecomposition may comprise two mimics of the same microRNA. In yet someother embodiments, the invention provides pharmaceutical compositionscomprising a therapeutically effective amount of three microRNA mimeticcompounds of the invention and a pharmaceutically acceptable carrier orexcipient. For instance, a pharmaceutical composition may comprise acombination of a miR-29a, a miR-29b and a miR-29c mimics.

Preferably, in the pharmaceutical compositions comprising at least twomicroRNA mimetic compounds according to the invention, the first and thesecond mimetic compounds or the first, second and the third mimeticcompounds are present in equimolar concentrations. Other mixing ratiossuch as about 1:2, 1:3, 1:4, 1:5, 1:2:1, 1:3:1, 1:4:1, 1:2:3, 1:2:4 arealso envisioned for preparing pharmaceutical compositions comprising atleast two of the miR-29a, miR-29b, and miR-29c mimetic compounds.

In some embodiments, one or more microRNA mimetic compounds of theinvention may be administered concurrently but in separate compositions,with concurrently referring to mimetic compounds given within a shortperiod, for instance, within about 5, 10, 20, or 30 minutes of eachother. In some other embodiments, miR-29a, miR-29b, and/or miR-29cmimetic compounds may be administered in separate compositions atdifferent times.

The invention also encompasses embodiments where additional therapeuticagents may be administered along with miR-29a, miR-29b, and/or miR-29cmimetic compounds. In one embodiment, the additional therapeutic agentis a second anti-fibrotic agent. The additional therapeutic agents maybe administered concurrently but in separate formulations orsequentially. In other embodiments, additional therapeutic agents may beadministered at different times prior to after administration ofmiR-29a, miR-29b, and/or miR-29c mimetic compounds. Where clinicalapplications are contemplated, pharmaceutical compositions will beprepared in a form appropriate for the intended application. Generally,this will entail preparing compositions that are essentially free ofpyrogens, as well as other impurities that could be harmful to humans oranimals.

Colloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, liposomes andexosomes, may be used as delivery vehicles for miR-29a, miR-29b, and/ormiR-29c mimetic compounds. In some embodiments, miR-29 mimics of thepresent invention may be formulated into liposome particles, which canthen be aerosolized for inhaled delivery.

Commercially available fat emulsions that are suitable for deliveringthe nucleic acids of the invention to target tissues includeIntralipid®, Liposyn®, Liposyn® II, Liposyn® III, Nutrilipid, and othersimilar lipid emulsions. A preferred colloidal system for use as adelivery vehicle in vivo is a liposome (i.e., an artificial membranevesicle). The preparation and use of such systems is well known in theart. Exemplary formulations are also disclosed in U.S. Pat. No.5,981,505; U.S. Pat. No. 6,217,900; U.S. Pat. No. 6,383,512; U.S. Pat.No. 5,783,565; U.S. Pat. No. 7,202,227; U.S. Pat. No. 6,379,965; U.S.Pat. No. 6,127,170; U.S. Pat. No. 5,837,533; U.S. Pat. No. 6,747,014;and WO03/093449, which are herein incorporated by reference in theirentireties.

In certain embodiments, liposomes used for delivery are amphotericliposomes such SMARTICLES® (Marina Biotech, Inc.) which are described indetail in U.S. Pre-grant Publication No. 20110076322. The surface chargeon the SMARTICLES® is fully reversible which make them particularlysuitable for the delivery of nucleic acids. SMARTICLES® can be deliveredvia injection, remain stable, and aggregate free and cross cellmembranes to deliver the nucleic acids.

One will generally desire to employ appropriate salts and buffers torender delivery vehicles stable and allow for uptake by target cells.Aqueous compositions of the present invention comprise an effectiveamount of the delivery vehicle comprising the miR-29 mimic (e.g.liposomes or other complexes) dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium. The phrases“pharmaceutically acceptable” or “pharmacologically acceptable” refersto molecular entities and compositions that do not produce adverse,allergic, or other untoward reactions when administered to an animal ora human. As used herein, “pharmaceutically acceptable carrier” includessolvents, buffers, solutions, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents and thelike acceptable for use in formulating pharmaceuticals, such aspharmaceuticals suitable for administration to humans. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active ingredients of the present invention, itsuse in therapeutic compositions is contemplated. Supplementary activeingredients also can be incorporated into the compositions, providedthey do not inactivate the polynucleotides of the compositions.

In one embodiment, pharmaceutical compositions of the invention areformulated for pulmonary, nasal, intranasal or ocular delivery and canbe in the form of powders, aqueous solutions, aqueous aerosols, nasaldrops, aerosols, and/or ocular drops. Solid formulations fornasal/intranasal administration may contain excipients such as lactoseor dextran. Liquid formulations for nasal/intranasal administration maybe aqueous or oily solutions for use in the form of aerosols, nasaldrops or metered spray. Formulations for pulmonary/nasal/intranasaladministration may also include surfactants such as, for example,glycocholic acid, cholic acid, taurocholic acid, ethocholic acid,deoxycholic acid, chenodeoxycholic acid, dehydrocholic acid,glycodeoxycholic acid, salts of these acids, and cyclodextrins.

In some embodiments, formulations for pulmonary/nasal/intranasaladministration via inhalation include, but are not limited to a drypowder formulation, a liposomal formulation, a nano-suspensionformulation, or a microsuspension formulation.

In some embodiments, pharmaceutical compositions forpulmonary/nasal/intranasal delivery are administered using an inhalationdevice. The term “inhalation device” refers to any device that iscapable of administering a miR-29 mimic composition to the respiratoryairways of the subject. Inhalation devices include devices such asmetered dose inhalers (MDIs), dry powder inhalers (DPIs), jetnebulizers, ultrasonic wave nebulizers, heat vaporizers, soft mistinhalers, thermal aerosol inhalers, electrohydrodynamic-based solutionmisting inhaler. Inhalation devices also include high efficiencynebulizers. In some embodiments, a nebulizer is a jet nebulizer, anultrasonic nebulizer, a pulsating membrane nebulizer, a nebulizercomprising a vibrating mesh or plate with multiple apertures, anebulizer comprising a vibration generator and an aqueous chamber, or anebulizer that uses controlled device features to assist inspiratoryflow of the aerosolized aqueous solution to the lungs of the subject.Nebulizers, metered dose inhalers, and soft mist inhalers deliverpharmaceuticals by forming an aerosol which includes droplet sizes thatcan easily be inhaled.

In some embodiments, a composition administered with a high efficiencynebulizer comprises one or more miR-29 mimics and pharmaceuticallyacceptable excipients or carriers such as purified water, mannitol,surfactants, and salts such as sodium chloride and sodium EDTA, etc.

The active compositions of the present invention may include classicpharmaceutical preparations. Administration of these compositionsaccording to the present invention may be via any common route so longas the target tissue is available via that route. This includes oral,nasal (e.g. inhalational), ocular, or buccal. Alternatively,administration may be by intravenous, intradermal, subcutaneous,intraocular or intramuscular injection, or by direct injection intopulmonary or cardiac tissue. Pharmaceutical compositions comprisingmiRNA mimics may also be administered by catheter systems or systemsthat isolate coronary circulation for delivering therapeutic agents tothe heart. Various catheter systems for delivering therapeutic agents tothe heart and coronary vasculature are known in the art. Somenon-limiting examples of catheter-based delivery methods or coronaryisolation methods suitable for use in the present invention aredisclosed in U.S. Pat. No. 6,416,510; U.S. Pat. No. 6,716,196; U.S. Pat.No. 6,953,466, WO 2005/082440, WO 2006/089340, U.S. Patent PublicationNo. 2007/0203445, U.S. Patent Publication No. 2006/0148742, and U.S.Patent Publication No. 2007/0060907, which are all herein incorporatedby reference in their entireties. Such compositions would normally beadministered as pharmaceutically acceptable compositions as describedherein.

In another embodiment of the invention, compositions comprising miR-29mimics as described herein may be formulated as a coating for a medicaldevice, such as a stent, balloon, or catheter. Particularly useful inmethods of treating cardiac fibrosis in a subject, the miR-29 mimics canbe used to coat a metal stent to produce a drug-eluting stent. Adrug-eluting stent is a scaffold that holds open narrowed or diseasedarteries and releases a compound to prevent cellular proliferationand/or inflammation. The mimetic compounds may be applied to a metalstent imbedded in a thin polymer for release of the agonists orinhibitors over time. Methods for device-based delivery and methods ofcoating devices are well known in the art, as are drug-eluting stentsand other implantable devices. See, e.g., U.S. Pat. Nos. 7,294,329,7,273,493, 7,247,313, 7,236,821, 7,232,573, 7,156,869, 7,144,422,7,105,018, 7,087,263, 7,083,642, 7,055,237, 7,041,127, 6,716,242, and6,589,286, and WO 2004/004602, which are herein incorporated byreference in their entireties. Thus, the present invention includes amedical device, such as a balloon, catheter, or stent, coated with amiR-29 mimic.

Sterile injectable solutions may be prepared by incorporating the activecompounds in an appropriate amount into a solvent along with any otheringredients (for example as enumerated above) as desired, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the desired otheringredients, e.g., as enumerated above.

The compositions of the present invention generally may be formulated ina neutral or salt form. Pharmaceutically-acceptable salts include, forexample, acid addition salts (formed with the free amino groups of theprotein) derived from inorganic acids (e.g., hydrochloric or phosphoricacids), or from organic acids (e.g., acetic, oxalic, tartaric, mandelic,and the like). Salts formed with the free carboxyl groups of the proteincan also be derived from inorganic bases (e.g., sodium, potassium,ammonium, calcium, or ferric hydroxides) or from organic bases (e.g.,isopropylamine, trimethylamine, histidine, procaine and the like).

Upon formulation, solutions are preferably administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective. The formulations may easily be administeredin a variety of dosage forms such as injectable solutions, drug releasecapsules, drug-eluting stents or other coated vascular devices, and thelike. For parenteral administration in an aqueous solution, for example,the solution generally is suitably buffered and the liquid diluent firstrendered isotonic for example with sufficient saline or glucose. Suchaqueous solutions may be used, for example, for intravenous,intramuscular, subcutaneous, intradermal, intraocular, andintraperitoneal administration.

This invention is further illustrated by the following additionalexamples that should not be construed as limiting. Those of skill in theart should, in light of the present disclosure, appreciate that manychanges can be made to the specific embodiments which are disclosed andstill obtain a like or similar result without departing from the spiritand scope of the invention.

All patent and non-patent documents referenced throughout thisdisclosure are incorporated by reference herein in their entirety forall purposes.

EXAMPLES Example 1: In Vitro Activity of miR-29 Mimic

To test for functional efficacy, a miR-29b mimic containing SEQ ID NO: 2as the first strand and SEQ ID NO: 1 as the second strand wastransfected into a mouse fibroblast cell line 3T3) and the effect onCollagen1a1 (Col1a1) 1a1) expression, a known direct target gene ofmiR-29 (van Rooij et al, 2008), was measured using qPCR. Increasingamount of miR-29b mimic showed a dose-dependent decrease in Col1a1,compared to untreated, mock transfected (without any oligo) ornon-targeting control (NTC) oligo treated cells, indicating the miR-29bmimic to be functional. A siRNA directly targeting Col1a1 was used as apositive control (FIG. 1B).

Example 2: In Vivo Distribution, Stability and Clearance of miR-29 Mimic

To explore the in vivo applicability and distribution of the miR-29mimic, mice were injected intravenously with 10, 50, 100, or 125 mg perkg of the miR-29b mimic containing SEQ ID NO: 2 as the first strand andSEQ ID NO: 1 as the second strand and sacrificed four days later.

Total RNA was isolated from cardiac tissue samples by using TRIzol®reagent (solution of phenol and guanidine isothiocynate) (Gibco/BRL).Northern blots to detect microRNAs were performed as previouslydescribed (van Rooij et al, 2008). A U6 probe served as a loadingcontrol (IDT). 10 μg of total RNA from the indicated tissues was loadedon 20% acrylamide denaturing gels and transferred to Zeta-probe GTgenomic blotting membranes (Bio-Rad) by electrophoresis. After transfer,the blots were cross-linked and baked at 80° C. for 1 hr. To maximizethe sensitivity of miRNA detection, oligonucleotide probes were labeledwith the Starfire Oligos Kit (IDT, Coralville, Iowa) and α-³²P dATP(Amersham or Perkin Elmer). Probes were hybridized to the membranesovernight at 39° C. in Rapid-hyb buffer (Amersham), after which theywere washed twice for 10 minutes at 39° C. with 0.5×SSC containing 0.1%SDS. The blots were exposed and quantified by PhosphorImager analysis(GE HealthCare Life Sciences) and a U6 probe served as a loading control(ABI). The intensity of the radioactive signal was used to quantify thefold change in expression using a phosphorimager and ImageQuant(Bio-Rad).

For real-time PCR analysis, RNA was extracted from cardiac tissue usingTrizol (Invitrogen) after which one to two μg RNA from each tissuesample was used to generate cDNA using Super Script II reversetranscriptase per manufacturer's specifications (Invitrogen). TaqmanMicroRNA assay (Applied Biosystems, ABI) was used to detect changes inmiRNAs or genes according the manufacturer's recommendations, using10-100 ng of total RNA. U6 was used a control for miRNA analysis andGAPDH was used as a control for gene analysis.

Northern blot analysis on multiple tissues indicated little to noincrease in miR-29b in kidney or liver samples compared to salinecontrol. Cardiac distribution was detected; however this appeared to bequite variable and spleen delivery could be observed at the highest doseonly. However, delivery to the lungs could be observed at all 3 of thehighest doses four days after injection (FIG. 1C). No effects on liverfunction (transaminase, ALT) were observed in the plasma, indicatingthat these miRNA mimics are well tolerated at these doses (FIG. 5).Real-time PCR demonstrated similar results with robust dose-dependentdistribution of the miR-29b mimic to the lung compared to salineinjected animals (FIG. 1D). Additionally, real-time PCR analysis ofmiR-29 targets showed no regulation at the mRNA level in the treatedanimals except for Col3a1 at the highest dose in the spleen (FIG. 6).This suggests that the target genes are either at steady-state innon-stressed animals and that mimics lower target genes when they areelevated, or that functional delivery was inadequate or insufficient.

To gain more insights into the in vivo stability of miRNA mimics, 125mpk of the miR-29b mimic was injected into mice and they were sacrificed1, 2, 4, or 7 days later. Robust presence of miR-29b mimic could bedetected by both Northern blot and real-time PCR analysis one day afterinjection in all tissues examined, however tissue clearance greatlydiffered thereafter (FIGS. 1E and F). Liver and kidney rapidly clearedmiR-29b mimic with minimal detection after day 1. Lung and spleendemonstrated the most pronounced detection of miR-29b mimic over time,which was sustained at least 4 days post-treatment (FIGS. 1E and 1F).The increase was specific for miR-29b without any effect on miR-29a andmiR-29c levels as measured by real-time PCR (FIG. 7). Also herereal-time PCR analysis of miR-29 targets showed no downregulation at themRNA level in non-stressed animals (FIG. 8).

Together these data indicate that unformulated miR-29b mimic canincrease the miRNA level with tissue-dependent clearance and deliveryefficiency, without any clear effect on gene expression under baselineconditions.

Example 3: miR-29b Mimic Blunts Bleomycin-Induced Pulmonary Fibrosis

Current treatments of tissue fibrosis mostly rely on targeting theinflammatory response; however these treatments are ultimatelyineffective in preventing progression of the disease, underscoring theneed for new mechanistic insights and therapeutic approaches (Friedmanet al, 2013). Recent studies indicate the involvement of miRNAs inpulmonary fibrosis (Pandit et al, 2011).

Due to the preferential lung distribution of the miR-29b mimic, thequestion of whether stress and subsequent induction of target geneexpression would allow for detectable changes in mRNA target genes anddownstream therapeutic effects in response to treatment with miR-29bmimic was explored. To test this, the bleomycin-induced model ofpulmonary fibrosis was used as previously described (Pandit et al,2010). Specifically, mice were anesthetized by placing them in a chamberhaving paper towels soaked with 40% isoflurane solution. 0.0375 U ofbleomycin (Hospira, Ill.) was administered intratracheally in 50 μl of0.9% saline. To determine the effect of miR-29b mimicry on earlyfibrosis, control (saline-treated) and bleomycin-treated mice wereinjected with 100 mg per kg of the miR-29b mimic containing SEQ ID NO: 2as the first strand and SEQ ID NO: 1 as the second strand, control mimicor a comparable volume of saline at two time-points: 3 and 10 days afterbleomycin treatment; the animals were sacrificed and lungs harvested atday 14. To determine the effect of miR-29b mimicry on establishedfibrosis, the miR-29b mimic was administered at days 10, 14 and 17 afterbleomycin or saline and the mice were sacrificed at day 21. In bothprotocols, the lungs were harvested for histological analysis,hydroxyproline assay and RNA extraction.

As expected, 14 days after bleomycin treatment, miR-29 levels werereduced, while miR-29b mimic treatment resulted in the increaseddetection of miR-29b levels compared to either control or salineinjected animals as measured by real-time PCR, albeit with a high levelof variation (FIG. 2A). To determine whether a similar decline in miR-29levels is observed in humans, sixteen lung tissue samples were obtainedfrom surgical remnants of biopsies or lungs explanted from patients withidiopathic pulmonary fibrosis (IPF) who underwent pulmonarytransplantation. Samples were obtained from University of PittsburghHealth Sciences Tissue Bank. A comparable decline in miR-29 levels wasobserved in pulmonary biopsies of patients with idiopathic pulmonaryfibrosis (IPF) compared to normal controls (FIG. 2B).

For histological examination, lung tissue sections (4 μm) were stainedwith Masson Trichrome (collagen/connective tissue), two slices peranimal, two animals per group. Immune staining was performed afterparaffin removal, hydration, and blocking, following the recommendationof the manufacturer (ABC detection system form Vector's lab, USA).Sections were incubated overnight at 4° C. with the primary antibody(Igf1, diluted 1:100 in PBS) and during 1 hour at room temperature withthe secondary antibodies (Invitrogen, USA). The sections werecounterstained with hematoxylin. The primary antibody was replaced bynonimmune serum for negative controls. Finally, sections were mountedwith mounting medium (DAKO, USA) and analyzed using a Nikon microscope.Histological analysis showed a clear and robust fibrotic andinflammatory response to bleomycin treatment, which was blunted bymiR-29b mimic treatment (FIG. 2C).

Additionally, lung hydroxyproline was analyzed for total collagencontent with hydroxyproline colorimetric assay kit from Biovision(Milpitas, Calif.) following manufacturer's instruction. Briefly, thelungs from control and experimental mice were dried until constantweight and hydrolyzed in 12N HCl for 3 hours at 120° C. The digestionsreacted with Chloramine T reagent and visualized in DMAB reagent. Theabsorbance was measured at 560 nm in a microplate reader. Data areexpressed as μg of hydroxyproline/right lung.

The hydroxyproline analysis indicated a significant increase followingbleomycin treatment in both saline and control treated groups, whilethere was no statistical difference in the miR-29 mimic treated groupbetween saline and bleomycin-treated mice, indicating that miR-29b mimictreatment blunts bleomycin-induced pulmonary fibrosis (FIG. 2D).

Innate immune effector signaling pathways act as important drivers ofmyofibroblast transdifferentiation by provoking fibrosis. To furthercharacterize the therapeutic effects of miR-29b mimic in the setting ofbleomycin-induced pulmonary fibrosis, bronchoalveolar lavage (BAL) wasperformed on these mice and cytokine levels were assessed using a humanCytokine/Chemokine Panel from Bio-Rad. The entire procedure wasperformed following manufacturer's instruction. Briefly, BALs werediluted five-fold and assay was performed in 96-well filter plates. Forthe detection step, samples were incubated for 30 min with streptavidinconjugated to R-phycoerythrin and analyzed in the Bio-Plex suspensionarray system (Bio-Rad). Raw data was analyzed using Bioplex Managersoftware 6.0 (Bio-Rad). The cytokine standards supplied by themanufacturer were used to calculate the concentrations of the samples.The analytes that were below the detection range were not included indate interpretation. Also, samples that had a particular analyte belowthe detection range were excluded while calculating the median value.

Significantly higher concentrations of IL-12, IL-4 and G-CSF weredetectable in BAL fluids from lungs from bleomycin-treated mice, whichwere reduced with miR-29b mimic (FIGS. 2E to 2G). Additionally, thebleomycin-induced elevation of detectable immune cells in BAL fluids wassignificantly reduced in the presence of miR-29b mimic (FIG. 2H),indicating an inhibitory effect on the immune response by miR-29b, whichis likely secondary to the antifibrotic-effect. To determine if miR-29mimicry has a direct effect on macrophages, miR-29b mimic or control wastransfected into macrophage cells, RAW 264.7, and the cell supernatantwas harvested at 24 and 48 hours after transfection. IFN-r, IL-1B, IL-2,IL-4, IL-5, IL-6, KC, IL-10, IL-12P70, and TNF-α were assessed, with nosignificant differences observed between miR-29b mimic and control (datanot shown). By real-time PCR analysis, there were no significantdifferences in Tgfb1, Ctgf, FGF 1, or PDGF expression; however, asignificant difference in Csf3, Igf1, and Kc expression was observed(FIG. 9 and data not shown).

Since it has been well validated that miR-29 functions through theregulation of many different extracellular matrix related genes (vanRooij & Olson, 2012), the regulation of a subset of these target geneswas confirmed. While a significant increase in Col1a1 and a trendincrease in Col3a1 expression were observed with bleomycin treatment inboth saline and control-treated groups, the detection of Col1a1 andCol3a1 was significantly blunted in the presence of miR-29b mimic in thebleomycin-treated mice (FIGS. 3A and 3B). Interestingly, the increase inIgf1 levels in BAL fluids following bleomycin treatment weresignificantly blunted in the presence of miR-29 mimic compared to bothsaline and control-treated mice (FIG. 3C). Furthermore,immunohistochemistry for Igf1 demonstrated robust reductions in Igf1after bleomycin in miR-29b mimic-treated groups compared to saline orcontrols (FIG. 3D).

After establishing that early (day 3 and day 10) miR-29 mimicry wassufficient to prevent bleomycin induced fibrosis, the ability of miR-29mimicry to affect established fibrosis was investigated. For thatpurpose, the miR-29b mimic administration was started at day 10 postbleomycin, and the doses were repeated at days 14 and 17, after whichthe lungs were harvested at day 21. Hydroxyproline assessment of theright lung showed a significant increase with bleomycin in both salineand control-treated lungs; however miR-29b mimic treatment blunted thiseffect (FIG. 4A). Furthermore, bleomycin treatment resulted insignificant increases in Col1a1 and Col3a1 expression, which was alsonormalized with miR-29b mimic treatment (FIGS. 4B and 4C). Histologicalassessment by trichrome staining corroborated this effect, wherebybleomycin induced significant fibrosis with saline or control treatmentwhich was blunted with miR-29b mimicry (FIG. 4D).

While it was believed that these observed effects were mediated throughregulation of collagen production from lung fibroblasts, the effectsfrom other collagen producing cells could not be ruled out. To addressthis issue, miR-29b mimic effects were assessed in vitro from differentlung cells, including primary fibroblasts from IPF patients and A549cells, a lung epithelial cell line. As expected, primary pulmonaryfibroblasts from IPF patients show an increase in Col1a1 and Col3a1 inresponse to TGF-α (FIGS. 4E and 4F). This effect was dose-dependentlyblunted with miR-29b mimic treatment at both 24 and 48 hours (FIGS. 4E,4F and data not shown). Similarly, A549 cells respond to TGF-α withrobust increases in Col1a1 and Col3a1 expression (FIGS. 4G and 4H).Again, miR-29b mimic treatment is able to block collagen induction, inboth TGF-α treated as well as baseline conditions (FIGS. 4G and 4H). Theeffects on collagen induction are much more robust in the A549 cellscompared to primary IPF cells. However, this is likely due to thealready high expression in primary fibroblasts from IPF patients.Additionally, miR-29 effects in the macrophage line, Thp-1, were alsoexamined, but no collagen expression could be observed in the cells,regardless of stimulation (data not shown). These data suggest miR-29bmimicry is able to blunt collagen-induced expression in fibroblasts andepithelial cells. These data are in agreement with a paper by Xiao etal., in which they showed that gene transfer of miR-29 using a SleepingBeauty-transposon system was capable of preventing and treatingbleomycin-induced pulmonary fibrosis (Xiao et al, 2012), furtherunderscoring the therapeutic potential for increasing miR-29.

The data described herein shows the feasibility of using microRNA mimicsto restore the function of lost or down-regulated miRNAs. However, it isimportant to note that because RISC incorporation is required forappropriate miRNA function, careful design of the structural features ofthe synthetic oligonucleotide mimics is required. In addition, the datain the present application shows that delivery to the appropriate celltype or tissue provides effective miRNA mimicry. For instance, in thecase of pulmonary fibrosis, direct delivery through the inhaled routeprovides better treatment efficacy compared to traditional routes ofadministration.

Example 4: miR-29b Mimic Blunts Extracellular Matrix Production in theSkin

In addition to organ fibrosis, a number of studies have shown a role formiR-29 in dermal fibrosis such as in hypertrophic scars and keloids, andin cutaneous and other forms of systemic sclerosis (scleroderma). Forexample, fibroblasts and skin sections obtained from patients withsystemic sclerosis showed a dramatic reduction in miR-29a levelscompared to healthy controls (Maurer et al 2010). When overexpressed insystemic sclerosis fibroblasts, miR-29a was able to robustly reduce theexpression of type I and III collagens, at both the RNA and proteinlevel. Conversely, inhibition of miR-29 in normal fibroblasts resultedin an increase in these collagens. Further, bleomycin-treated skinshowed a significant reduction in miR-29 as well, suggesting miR-29down-regulation is broadly applicable in multiple indications offibrosis (Maurer et al 2010). These results were further validated inanother study where miR-29a transfection in healthy control dermalfibroblasts significantly down-regulated collagen expression.Additionally, miR-29a overexpression in dermal fibroblasts decreasedsecreted TIMP-1 and increased collagen gel degradation. These resultswere validated in fibroblasts from patients with systemic sclerosis aswell (Ciechomska et al 2014). Collectively, these studies point tomiR-29 mimicry as a therapeutic potential in local forms of fibrosis,including dermal fibrosis and systemic sclerosis.

To determine the effect of miR-29 agonism or antagonism in skin, a mouseincisional wound model was used. Specifically, male C57BL/6 mice wereanesthetized with 2-5% inhaled isofluorane using an inhalation chamberand were maintained under 1% isofluorane on a nose cone for allprocedures. Buprenorphine (0.1 mg/kg) was used as an analgesic for allsurgical procedures. Animals were anesthetized, depilated by shaving andadministration of Nair hair removal cream, and the skin site wasprepared for incision by betadine and alcohol surgical scrub. One to two1 cm long skin incisions were made on the backs of the mice. Incisionswere closed with 2-5 interrupted sutures and were covered with Tegadermtransparent semi-occlusive dressing (3M). Mice with or withoutincisional skin wounds were treated with intradermal injection withvehicle (PBS), 100 nmol miR-29b mimic (comprising SEQ ID NO: 2 as thefirst strand and SEQ ID NO: 1 as the second strand) or 100 nmolantimiR-29, 3 days after incisional wound creation, by intradermalinjection of oligonucleotide compounds or vehicle (PBS) in two 50 μLvolumes located on either side of the incision midline, or by injectionof a single 100 μL volume into adjacent, unwounded skin. The antimiR-29has the sequence of5′-lGs.dAs.dTs.dTs.lTs.lCs.dAslAs.dAs.lTs.lGs.dGs.lTs.dGs.lCs.lTs-3′(SEQ ID NO: 36) where “l” represents a locked nucleotide, “d” representsa deoxyribonucleotides and “s” represents a phosphorothioate linkage.Mice were sacrificed 24 hours after administration of oligonucleotidecompounds and skin at the treatment site(s) was harvested and snapfrozen in liquid nitrogen for analysis of mRNA expression.

Total RNA was extracted from skin using Trizol (Invitrogen) andreal-time PCR analysis was performed as described above except thatGAPDH, B2M, HPRT1 and PPIB were used as controls for gene analysis ofskin samples.

For microarray analysis, one μg total RNA per sample was sent to MOgenefor microarray analysis as compared to a mouse universal reference RNA(Agilent) on an Agilent array (Mouse GE (V2), 4×44k-026655). Analysiswas performed using ArrayStudio and heatmaps were generated using the Rsoftware program.

Using microarray analysis, positively and negatively regulated geneswere identified relative to PBS controls. 228 genes were reciprocallyregulated (p<0.05 or fold change >1.5) between the miR-29b mimic andantimiR-29 (FIG. 10 and Table 5). These miR-29 regulated genes and theirorthologs in other species may be utilized as translational biomarkersto indicate response to treatment with a miR-29 agonist or miR-29antagonist.

TABLE 5 miR-29b mimic antimiR-29 Gene Fold change p value Fold change pvalue Cytl1 −3.21 0.000 1.53 0.034 Col3a1 −2.68 0.074 1.61 0.352 Col1a1−2.43 0.051 2.16 0.081 Col1a2 −2.38 0.028 2.26 0.036 Fstl1 −2.25 0.0152.15 0.019 Col5a2 −2.19 0.005 1.95 0.012 4930543N07Rik −2.13 0.005 3.100.000 Faim2 −2.13 0.083 2.42 0.049 Tmem213 −2.06 0.047 2.39 0.022 Tgfb3−1.99 0.002 −1.29 0.145 Lzts1 −1.97 0.001 1.43 0.017 Eln −1.95 0.0041.14 0.472 Slc5a2 −1.94 0.018 2.09 0.010 Tnp2 −1.93 0.073 4.05 0.002Olfr1336 −1.87 0.001 1.75 0.002 Tdrd9 −1.82 0.113 2.85 0.014 Gm6602−1.81 0.194 2.83 0.038 F830016B08Rik −1.78 0.098 2.16 0.037 Myo3b −1.780.034 1.82 0.030 Colec11 −1.77 0.010 1.80 0.009 Gm10428 −1.75 0.010 1.940.005 Vmn1r65 −1.70 0.042 3.21 0.001 Olfr67 −1.69 0.003 1.37 0.034 Bsnd−1.69 0.107 2.05 0.038 Slc10a5 −1.69 0.038 1.65 0.045 Defa26 −1.68 0.0762.06 0.022 Serpinh1 −1.68 0.124 2.01 0.049 Gm5606 −1.67 0.018 1.77 0.011Wfdc11 −1.67 0.252 2.64 0.048 Gimap7 −1.65 0.091 1.92 0.036 Nedd4l −1.640.001 1.34 0.012 Cacna1g −1.63 0.094 1.81 0.049 Prickle1 −1.63 0.0011.25 0.036 4931415C17Rik −1.62 0.182 2.94 0.012 D730005E14Rik −1.610.156 2.30 0.025 Ccr10 −1.60 0.014 1.50 0.028 Gm22 −1.60 0.003 2.560.000 Ngp −1.60 0.075 1.83 0.030 Ascl1 −1.60 0.026 1.78 0.010 Tgfb2−1.60 0.016 1.08 0.618 Cyp2c29 −1.59 0.009 1.42 0.030 Gm5797 −1.59 0.0111.49 0.021 Col5a3 −1.59 0.035 1.05 0.779 A730093L10Rik −1.59 0.060 1.690.039 Fkbp10 −1.57 0.027 1.57 0.028 Mfap2 −1.56 0.032 1.83 0.008 Gm5485−1.55 0.064 5.68 0.000 Slamf9 −1.54 0.204 2.08 0.048 Mab21l3 −1.54 0.0051.53 0.006 Fam57b −1.53 0.008 1.50 0.010 Pcolce −1.53 0.091 1.62 0.060Gm6760 −1.52 0.215 2.26 0.031 Gng13 −1.51 0.073 2.07 0.006 4933404M02Rik−1.50 0.361 3.49 0.018 C1qtnf6 −1.50 0.032 1.50 0.031 Tmem119 −1.490.019 1.46 0.023 Ubtd2 −1.49 0.001 1.48 0.001 Rasl11b −1.48 0.045 1.770.009 Nr5a2 −1.47 0.011 1.36 0.031 Gm3727 −1.46 0.018 1.43 0.024 Gprasp2−1.46 0.044 1.93 0.003 Syt10 −1.45 0.015 2.08 0.000 Otog −1.44 0.0361.65 0.009 Bdh2 −1.43 0.011 1.81 0.001 Sema3b −1.42 0.045 1.56 0.017Al118078 −1.42 0.032 1.69 0.005 Npc1l1 −1.41 0.032 1.40 0.036 Dnmt3a−1.41 0.015 1.30 0.046 Cxcr6 −1.40 0.024 1.99 0.000 Sh3pxd2a −1.37 0.0441.42 0.029 Scarf2 −1.35 0.004 1.67 0.000 LOC100862627 −1.34 0.006 1.450.002 Selm −1.34 0.015 1.42 0.006 Col11a1 −1.34 0.509 −1.02 0.958Slc12a5 −1.34 0.007 1.29 0.013 Pex11c −1.33 0.000 1.24 0.002 Gpr176−1.32 0.041 1.68 0.002 Qprt −1.32 0.037 1.29 0.050 Phldb2 −1.31 0.0421.52 0.006 Prl2c1 −1.31 0.007 1.37 0.003 Rab39 −1.30 0.018 1.61 0.001Dact3 −1.30 0.003 1.60 0.000 Dlx3 −1.26 0.010 1.19 0.037 Sepw1 −1.250.021 1.26 0.017 Socs7 −1.25 0.019 1.22 0.030 Maged1 −1.24 0.008 1.260.006 Ckb −1.22 0.035 1.24 0.026 Mmp2 −1.21 0.288 1.55 0.031 Nfatc4−1.20 0.043 1.21 0.039 Gm13623 −1.20 0.032 1.51 0.000 Trp53i13 −1.190.025 1.32 0.002 Lysmd4 −1.17 0.015 1.23 0.004 Polr2m −1.17 0.000 1.120.002 Pkd1 −1.17 0.041 1.17 0.035 Zdhhc1 −1.16 0.034 1.25 0.005 Nlgn2−1.15 0.043 1.22 0.009 Gm9223 −1.15 0.005 1.30 0.000 Tox4 −1.14 0.0161.16 0.007 Josd1 −1.10 0.039 1.14 0.009 Trip12 −1.10 0.017 1.28 0.000Bet1l −1.10 0.020 1.24 0.000 Scaf1 −1.09 0.039 1.16 0.003 Dynlrb1 −1.070.015 1.07 0.024 Fam195b −1.07 0.001 1.24 0.000 Smarca5 1.05 0.013 −1.050.023 Rae1 1.07 0.016 −1.17 0.000 Nhp2l1 1.07 0.013 −1.23 0.000 Pin1-ps11.07 0.038 −1.13 0.003 Ppp1r7 1.08 0.011 −1.16 0.000 Lars 1.08 0.012−1.11 0.002 Wdsub1 1.09 0.031 −1.13 0.006 Fam120a 1.09 0.043 −1.10 0.0323010027C24Rik 1.10 0.032 −1.15 0.005 Eif4a3 1.10 0.021 −1.14 0.004 Vprbp1.10 0.042 −1.38 0.000 Naa20 1.10 0.008 −1.07 0.037 Smu1 1.11 0.006−1.17 0.001 Tmed10 1.11 0.004 −1.06 0.039 Dus1l 1.11 0.033 −1.16 0.006Ecd 1.11 0.026 −1.24 0.001 Naa10 1.11 0.015 −1.10 0.026 Ddx18 1.11 0.011−1.11 0.014 Btbd9 1.11 0.002 −1.06 0.037 Ubap2l 1.11 0.008 −1.11 0.008Pnkp 1.12 0.022 −1.11 0.023 Parl 1.12 0.022 −1.25 0.001 Tle4 1.12 0.011−1.20 0.001 Wbp11 1.12 0.027 −1.12 0.026 Nek4 1.12 0.027 −1.26 0.001Poc5 1.13 0.039 −1.12 0.046 Uchl5 1.13 0.002 −1.28 0.000 Recql 1.130.006 −1.09 0.030 Psmd3 1.13 0.011 −1.10 0.028 Asna1 1.13 0.033 −1.310.000 Polr1e 1.13 0.048 −1.20 0.009 Csrp2bp 1.13 0.023 −1.18 0.006 Parp11.13 0.049 −1.14 0.043 Abi1 1.13 0.003 −1.48 0.000 Tubgcp2 1.14 0.006−1.47 0.000 Reps1 1.14 0.007 −1.16 0.003 Mon2 1.14 0.023 −1.13 0.032Seh1l 1.14 0.034 −1.16 0.021 Mri1 1.14 0.000 −1.13 0.000 Ddx20 1.140.002 −2.18 0.000 Nup133 1.14 0.001 −1.09 0.006 Ubr7 1.15 0.006 −1.100.033 Fam32a 1.15 0.009 −1.16 0.007 Cct2 1.15 0.003 −1.26 0.000 Actl6a1.15 0.027 −1.15 0.024 Snrpb 1.15 0.012 −1.21 0.002 Ino80 1.15 0.032−1.14 0.038 1500002O20Rik 1.15 0.025 −1.28 0.001 Anxa7 1.15 0.008 −1.180.003 Wdr74 1.15 0.035 −1.32 0.001 Mrps27 1.16 0.015 −1.22 0.003Trnau1ap 1.16 0.007 −1.18 0.004 Usp39 1.16 0.031 −1.19 0.015 Mbd2 1.170.010 −1.12 0.043 Akap10 1.17 0.016 −1.35 0.000 Rps19bp1 1.17 0.031−1.23 0.008 Faf1 1.17 0.026 −1.25 0.004 Wdr55 1.17 0.002 −1.15 0.005Gorasp2 1.17 0.049 −1.20 0.027 Nfe2l2 1.17 0.011 −1.23 0.002 Nup54 1.170.048 −1.70 0.000 Med6 1.17 0.001 −1.17 0.002 Mapkap1 1.17 0.034 −1.190.026 Nsmce2 1.18 0.002 −1.09 0.043 Nsun2 1.18 0.011 −1.17 0.016 Map3k31.19 0.030 −1.28 0.005 Stat6 1.19 0.021 −1.40 0.001 Yrdc 1.19 0.008−1.25 0.002 Ap1m1 1.19 0.002 −1.15 0.005 Ccdc51 1.19 0.013 −1.17 0.019Gins4 1.19 0.012 −1.24 0.004 Tmem165 1.19 0.027 −1.21 0.018 Txnl1 1.190.031 −1.71 0.000 Zfp608 1.19 0.000 −1.30 0.000 Mphosph10 1.19 0.019−1.29 0.003 Spp1 1.20 0.014 −1.16 0.029 Wdr43 1.20 0.014 −1.16 0.028Atpbd4 1.20 0.004 −1.11 0.044 Pafah1b2 1.20 0.050 −1.20 0.049 Exosc81.20 0.027 −1.21 0.021 Nop14 1.20 0.003 −1.48 0.000 Nop16 1.20 0.038−1.22 0.030 Pdcd6ip 1.20 0.011 −1.90 0.000 Cbl 1.21 0.035 −1.83 0.000Pcif1 1.21 0.020 −1.24 0.011 Rbm14 1.21 0.045 −1.22 0.036 Epb4.1l5 1.210.049 −1.38 0.004 Mtmr10 1.21 0.041 −1.48 0.001 Ttf2 1.21 0.030 −1.420.001 Cenpo 1.22 0.009 −1.31 0.002 Rreb1 1.22 0.049 −1.84 0.000 Depdc51.22 0.002 −1.39 0.000 Umps 1.23 0.012 −1.16 0.046 Zfp52 1.23 0.039−1.55 0.001 BB070754 1.24 0.017 −1.28 0.010 Gnl3 1.24 0.036 −1.34 0.010Rbbp5 1.25 0.003 −1.34 0.001 Fam178a 1.26 0.048 −1.35 0.015 Etv5 1.270.035 −1.55 0.002 Gins1 1.27 0.034 −1.32 0.019 Lbr 1.28 0.002 −1.450.000 Gm5039 1.29 0.030 −1.57 0.002 Pgam1 1.29 0.027 −1.26 0.036 Atg71.29 0.005 −1.24 0.011 9030425P06Rik 1.30 0.005 −1.34 0.003 Lyst 1.300.028 −2.43 0.000 Rgs19 1.31 0.003 −1.24 0.012 Numb 1.31 0.001 −1.630.000 Snx27 1.32 0.014 −2.48 0.000 Rnf130 1.32 0.021 −1.31 0.026 Pias31.33 0.014 −1.90 0.000 Pqlc3 1.33 0.009 −1.30 0.013 Chka 1.33 0.005−1.44 0.001 A430105D02Rik 1.37 0.003 −3.13 0.000 Sdc4 1.38 0.004 −2.050.000 Rbm3 1.39 0.002 −1.56 0.000 5830468K08Rik 1.41 0.044 −1.89 0.002Clcn5 1.41 0.026 −1.49 0.014 Fam65b 1.41 0.049 −1.50 0.026 Tgfa 1.460.004 −1.34 0.015 Fgd4 1.48 0.000 −1.18 0.036 3930401B19Rik 1.57 0.020−1.46 0.043 Itga3 1.57 0.031 −1.63 0.024 2410137M14Rik 1.63 0.040 −1.640.039 Egr4 2.02 0.024 −1.86 0.040 Olfr663 2.06 0.022 −2.08 0.020

DAVID analysis (NCBI) of functional terms that are enriched in the twogroups are presented in FIG. 10B. Not surprisingly, the Gene Ontology(GO) terms of Extracellular Matrix, (Skin) Function, Adhesion/CellSignaling and Cell Differentiation/Apoptosis are the top negativelyregulated pathways following miR-29b mimic treatment. Cellular (Nuclear)Structure and RNA Processing are the top positively regulated pathwaysfollowing miR-29b mimic treatment.

To further confirm the effect of miR-29b mimic treatment in the skin,mice with acute incisional wounds were treated with intradermalinjection of PBS, 20, 50, or 100 nmol of miR-29b mimic (comprising SEQID NO: 2 as the first strand and SEQ ID NO: 1 as the second strand).Quantitative reverse-transcriptase PCR analysis was performed on 24genes identified as being direct or indirect targets of miR-29modulation in the skin (19 repressed by miR-29b mimic and upregulated byantimiR-29, 5 upregulated by miR-29b mimic and repressed by antimiR-29).Extracellular matrix genes (collagens, ELN, etc.) and others involved inthe fibrotic process (e.g. MMP2, TGFB2) were shown to be repressed bymiR-29b mimic treatment in the skin (FIG. 11A), whereas selected cellsurface receptors (ITGA3, LBR, NUMB, SDC4) and factors associated withreceptor endocytosis (SNX27) were shown to be increased with miR-29bmimic treatment in the skin (FIG. 11B). These studies indicate that themiR-29b mimic is active when treated locally in the skin, and suggeststhat in addition to its effect on organ fibrosis, miR-29 mimicry couldbe an effective therapy for cutaneous fibrosis of various etiologies.These studies also identify the above-mentioned genes as translationalbiomarkers whose expression correlates with the activity of a miR-29bmimic in the skin. These translational biomarkers can be utilized in aclinical trial, testing the safety and efficacy of miR-29b mimics innormal healthy volunteers and patients with cutaneous scleroderma.

Example 5: Activity of miR-29 Mimics and Effects of NucleotideModifications

To determine the efficacy of miR-29a, b, and c mimics in regulating theexpression of target genes, different miR-29a, b and c mimics weretransfected into IMR-90 human lung fibroblasts at a concentration of 10nM, and collagen expression was measured by quantitative RT-PCR. Thesestudies demonstrate that a miR-29a mimic comprising SEQ ID NO: 27 as thefirst strand and SEQ ID NO: 5 as the second strand and a miR-29b mimiccomprising SEQ ID NO: 19 as the first strand and SEQ ID NO: 1 as thesecond strand are the most effective at repressing expression ofmultiple collagen genes, whereas a miR-29c mimic comprising SEQ ID NO:35 as the first strand and SEQ ID NO: 24 as the second strand is lesseffective (FIG. 12). These effects may be cell-type or target genespecific, but indicate that there are indeed differences in the abilityof the three mimics to repress extracellular matrix gene expression.

In the same experiment, the effect of nucleotide modifications on theefficacy of miR-29b mimics was also tested. These studies indicate thatthe miR-29b mimic containing SEQ ID NO: 19 as the first strand and SEQID NO: 1 as the second strand performs similarly to a miR-29b mimiccomprising SEQ ID NO: 19 as the first strand and SEQ ID NO: 30 as thesecond strand which has the same first and second sequence and pattern,but a different chemical linker between the sense (passenger) strand andcholesterol. A checkerboard pattern of 2′ 0-Methyl modifications makesmiR-29 mimics (a mimic comprising SEQ ID NO: 31 as the first strand andSEQ ID NO: 28 as the second strand and a mimic comprising SEQ ID NO: 32as the first strand and SEQ ID NO: 29 as the second strand) completelyineffective. Modifications of all C and U residues on the antisense(first/guide) strand (a mimic comprising SEQ ID NO: 33 as the firststrand and SEQ ID NO: 1 as the second strand) partially reduces themimic activity. Similarly, removal of the 3′ overhang on the antisensestrand (a mimic comprising SEQ ID NO: 24 as the first strand and SEQ IDNO: 1 as the second strand) partially reduces the mimic activity. SeeFIG. 12.

These studies have allowed the stratification of miR-29 mimic compoundson the basis of in vitro activity in a specific cell line (IMR-90) andusing particular target genes (COL1A1, COL3A1, COL4A5) as the readoutfor efficacy, independent on compound uptake, and can be used as thebasis for selecting compounds to test via passive delivery in vitro orto test in vivo.

Example 6: In Vivo Activity of miR-29b Mimics with Linker Modifications

Mice with incisional wounds were treated with 20 nmol of various miR-29bmimics that differ only in the linkage between the cholesterol moietyand the second/sense strand. The mimetic compound that contained a sixcarbon linker between cholesterol and the sense strand (the mimiccomprising SEQ ID NO: 19 as the first strand and SEQ ID NO: 1 as thesecond strand) and the compound that contained the same six carbonlinker between cholesterol and the sense strand but connected through acleavable moiety (dT.dT) (SEQ ID NO: 19/SEQ ID NO: 15) showed similaractivity in repressing target genes (FIG. 13). N/S in FIG. 13 representsno significant difference was observed in the activities of the miR-29bmimic comprising SEQ ID NO: 19 as the first strand and SEQ ID NO: 1 asthe second strand and the mimic comprising SEQ ID NO: 19 as the firststrand and SEQ ID NO: 15 as the second strand. miR-29b mimics containinga nine carbon linker (SEQ ID NO: 19/SEQ ID NO: 17) and a linker at the5′ end (SEQ ID NO: 19/SEQ ID NO: 16) were not effective in repressingtarget genes (FIG. 13).

Example 7: Effect of 5′ Phosphorylation on the Activity of miR-29bMimics

RAB-9 skin fibroblast cells (ATCC CRL-1414) were transfected withvarying concentrations of miR-29b mimics with (the mimic containing SEQID NO: 2 as the first strand and SEQ ID NO: 1 as the second strand) andwithout (the mimic containing SEQ ID NO: 19 as the first strand and SEQID NO: 1 as the second strand) 5′ phosphorylation on the antisensestrand. No significant differences in target gene repression as measuredby Col1a1, Col1a2 or Col3a1 expression was observed in the activity ofthe two mimics (represented as N/S in FIG. 14). Both miR-29b mimicssignificantly (p<0.0001) repressed the expression of target genescompared to vehicle, mock transfection or control mimic treatment. SeeFIG. 14.

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The invention claimed is:
 1. A miR-29 mimetic compound comprising: afirst strand that is at least 23 ribonucleotides, comprising a maturemiR-29b sequence, wherein the first strand comprises a sequence selectedfrom SEQ ID NO: 2, 18-21, and 31-34 as provided in Table 2; and a secondstrand of about 22 to about 25 ribonucleotides, comprising a sequencethat is substantially complementary to the first strand and having atleast one modified nucleotide, wherein the first strand has a 3′nucleotide overhang relative to the second strand.
 2. The miR-29 mimeticcompound of claim 1 wherein the first strand has one or more 2′ fluoronucleotides.
 3. The miR-29 mimetic compound of claim 1, wherein the atleast one modified nucleotide in the second strand is a 2′-O-methylmodified nucleotide.
 4. The miR-29 mimetic compound of claim 1, whereinthe second strand has 1, 2, or 3 mismatches relative to the firststrand.
 5. The miR-29 mimetic compound of claim 4, wherein the secondstrand contains mismatches from the 3′ end at positions 4, 13, and/or 16relative to the first strand.
 6. The miR-29 mimetic compound of claim 1wherein the second strand is linked to a cholesterol molecule at its 3′or 5′ terminus.
 7. The miR-29 mimetic compound of claim 6, wherein thecholesterol molecule is linked to the second strand through at least asix carbon linker.
 8. The miR-29 mimetic compound of claim 1, whereinthe second strand comprises a sequence selected from SEQ ID NO: 1, 8-10,13-17, and 28-30.
 9. The miR-29 mimetic compound of claim 8, wherein thefirst strand comprises the sequence of SEQ ID NO: 19 and the secondstrand comprises the sequence of SEQ ID NO:
 1. 10. The miR-29 mimeticcompound of claim 8, wherein the first strand comprises the sequence ofSEQ ID NO: 19 and the second strand comprises the sequence of SEQ ID NO:15.
 11. A pharmaceutical composition comprising an effective amount ofthe miR-29 mimetic compound of claim 1, or a pharmaceutically-acceptablesalt thereof, and a pharmaceutically-acceptable carrier or diluent. 12.The pharmaceutical composition of claim 11, wherein the composition isan inhalation composition.
 13. The miR-29 mimetic compound of claim 1,wherein the first strand comprises:5′-fU.rA.rG.fC.rA.fC.fC.rA.fU.fU.fU.rG.rA.rA.rA.fU.fC.rA.rG.fU.rG.fU.fUs.rUs.rU-3′(SEQID NO: 19); and wherein the second strand comprises:5′-mA.mA.mC.rA.mC.mU.rG.mU.mU.mU.rA.mC.rA.rA.rA.mU.rG.rG.mU.mC.mC.mU.rA.cho16-3′(SEQ ID NO: 1).
 14. The miR-29 mimetic compound of claim 1, wherein thefirst strand comprises:5′-fU.rA.rG.fC.rA.fG.fC.rA.fU.fU.fU.rG.rA.rA.rA.fU.fC.rA.rG.fU.rG.fU.fUs.rUs.rU-3′(SEQID NO: 19); and wherein the second strand comprises:5′-mA.mA.mC.rA.mC.mU.rG.mU.mU.mU.rA.mC.rA.rA.rA.mU.rG.rG.mU.mC.mC.mU.rA.dT.dT.cho16-3′(SEQ ID NO 15).
 15. The miR-29 mimetic compound of claim 1, wherein thefirst strand comprises the sequence of SEQ ID NO:
 19. 16. The miR-29mimetic compound of claim 1, wherein the second strand comprises thesequence of SEQ ID NO:
 1. 17. The miR-29 mimetic compound of claim 1,wherein the second strand comprises the sequence of SEQ ID NO:
 15. 18.The miR-29 mimetic compound of claim 1, wherein the first strandcomprises:5′-fU.rA.rG.fC.rA.fC.fC.rA.fU.fU.fU.rG.rA.rA.rA.fU.fC.rA.rG.fU.rG.fU.fUs.rUs.rU-3′(SEQID NO: 19).
 19. The miR-29 mimetic compound of claim 1, wherein thesecond strand comprises:5′mA.mA.mC.rA.mC.mU.rG.mU.mU.mU.rA.mC.rA.rA.rA.mU.rG.rG.mU.mC.mC.mU.rA.cho16-3′(SEQ ID NO: 1).
 20. The miR-29 mimetic compound of claim 1, wherein thesecond strand comprises:5′-mA.mA.mC.rA.mC.mU.rG.mU.mU.mU.rA.mC.rA.rA.rA.mU.rG.rG.mU.mC.mC.mU.rA.dT.dT.cho16-3′(SEQID NO 15).
 21. A miR-29 mimetic compound comprising: a first strand ofabout 23 to about 26 ribonucleotides comprising a mature miR-29bsequence; and a second strand that is at least 23 ribonucleotides,comprising a sequence that is substantially complementary to the firststrand and having at least one modified nucleotide, wherein the firststrand has a 3′ nucleotide overhang relative to the second strand, andwherein the second strand comprises a sequence selected from SEQ ID NO:1, 8-10, 13-17, and 28-30 as provided in Table
 2. 22. The miR-29 mimeticcompound of claim 21 wherein the first strand has one or more 2′ fluoronucleotides.
 23. The miR-29 mimetic compound of claim 21, wherein thenucleotides comprising the 3′ overhang in the first strand are linked byphosphorothioate linkages.
 24. The miR-29 mimetic compound of claim 21,wherein the first strand comprises a sequence selected from SEQ ID NO:2, 18-21, and 31-34.
 25. The miR-29 mimetic compound of claim 24,wherein the first strand comprises the sequence of SEQ ID NO:
 19. 26.The miR-29 mimetic compound of claim 21, wherein the second strandcomprises the sequence of SEQ ID NO:
 1. 27. The miR-29 mimetic compoundof claim 21, wherein the second strand comprises the sequence of SEQ IDNO:
 15. 28. The miR-29 mimetic compound of claim 21, wherein the firststrand comprises:5′-fU.rA.rG.fC.rA.fC.fC.rA.fU.fU.fU.rG.rA.rA.rA.fU.fC.rA.rG.fU.rG.fU.fUs.rUs.rU-3′(SEQID NO: 19).
 29. The miR-29 mimetic compound of claim 21, wherein thesecond strand comprises:5′mA.mA.mC.rA.mC.mU.rG.mU.mU.mU.rA.mC.rA.rA.rA.mU.rG.rG.mU.mC.mC.mU.rA.cho16-3′(SEQ ID NO: 1).
 30. The miR-29 mimetic compound of claim 21, wherein thesecond strand comprises:5′-mA.mA.mCsA.mC.mUrGmU.mU.mU.rA.mC.rA.rA.rA.mUrG.rG.mUmC.mC.mUrA.dT.dT.cho16-3′(SEQID NO 15).
 31. A pharmaceutical composition comprising an effectiveamount of the miR-29 mimetic compound of claim 21, or apharmaceutically-acceptable salt thereof, and apharmaceutically-acceptable carrier or diluent.
 32. The pharmaceuticalcomposition of claim 31, wherein the composition is an inhalationcomposition.